Pancreatic endocrine progenitor cell therapies for the treatment of obesity and type 2 diabetes (t2d)

ABSTRACT

Provided herein are therapies, and methods using that therapy, in the treatment of one or more of Type 2 diabetes (T2D), obesity, glucose intolerance and insulin resistance or to control weight gain in subjects. In particular, the subject may be candidates for treatment with one or more small molecule anti-diabetic drugs and the therapy may include implanting a population of pancreatic endocrine progenitor cells into the subject, where the cells are allowed to mature in vivo to produce a population.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/131,540 filed on 11 Mar. 2015, entitled “THERAPYFOR THE TREATMENT OF OBESITY”.

TECHNICAL FIELD

The present invention relates to methods for treating metabolicdisorders, medical conditions and associated pathological conditions insubjects. In particular, the invention relates to the use of cellsresulting from the differentiation of pluripotent stem cells, alone orin combination with anti-diabetic medications, to achieve weight loss,improvements in glucose tolerance and/or enhanced insulin sensitivity insubjects.

BACKGROUND

Obesity is quickly becoming a global epidemic, which epidemic crossesall age and socio-economic groups. The number of overweight and obesepeople worldwide has risen from 857 million in 1980 to 2.1 billion in2013 (Ng, et al., Global, Regional & National Prevalence of Overweightand Obesity in Children and Adults During 1980-2013: A SystematicAnalysis for the Global Burden of Disease Study, Lancet (2014)).Additionally, obesity is known to be a major risk factor for thedevelopment of a number of diseases including Type 2 Diabetes or Type 2diabetes mellitus (T2D).

The International Diabetes Federation estimates that approximately 380million people worldwide have diabetes, up to 95% of which suffer fromT2D. In T2D, the body fails to properly use insulin, or is insulinresistant. T2D is also generally characterized by hyperglycemia, insulinresistance and low insulin levels. T2D is thought to be primarily due toobesity and lack of exercise in people who are genetically predisposed.

Diet, exercise and weight control are the cornerstones of managing T2D.However, drug therapy may be required in which one or more drugs areused to control blood sugar levels. Current medications for treatment ofT2D are oral medications including meglitinides, sulfonylureas,dipeptidyl-peptidase 4 (“DPP-4”) inhibitors, biguanides,thiazolidinediones, alpha-glucosidase inhibitors, and sodium-glucosetransporter 2 (“SGLT2”) inhibitors. Additionally, injectablemedications, such as amylin mimetics and incretin mimetics are used totreat T2D.

A second type of diabetes mellitus, Type 1 (T1D), is a chronic conditionin which little or no insulin is produced by the pancreas. Historically,T1D was treated with insulin administration in addition to the controlof diet, exercise and weight. However, more recently treatment hasincluded the transplantation of islets of Langerhans, which treatmentsuffers from a shortage of transplantable islets of Langerhans. Thus,even more recently, treatment development focused on developing sourcesof insulin-producing cells appropriate for engraftment. One suchapproach is the generation of insulin-producing cells from pluripotentstem cells, such as embryonic stem cells.

The production of enriched cultures of human embryonic stem cell-deriveddefinitive endoderm and the further differentiation of such cells intopancreatic endocrine precursor cells is known (for example,US2009/0170198; Rezania, A. et al. Diabetes 2012; Rezania, A. et al.Stem Cells 2013; Bruin, J. E. et al. 2013; Bruin, J. E. et al. Stem CellResearch 2014). Published patent application, US2012/0039955, alsodescribes a decrease in blood sugar in SCID mice with streptozotocin(STZ) induced T1D like state following transplantation of a populationof encapsulated pancreatic endocrine precursor cells. It is also knownthat subsequent transplant of the pancreatic endocrine precursor cellsinto a body allows for still further differentiation into functionalpancreatic endocrine cells.

SUMMARY

The present invention is based, in part, on the surprising discoverythat a combination therapy of pancreatic endocrine precursor cells(Stage 4 cells) and a small molecule anti-diabetic drug was moreeffective in high-fat diet (HFD) fed mice than either small moleculeanti-diabetic drugs or progenitor cell transplants alone. Moreover,surprisingly neither HFDs nor anti-diabetic drugs impacted the abilityof human embryonic stem cell (hESC)-derived cells to mature in vivo andappropriately secrete insulin in response to glucose. The environment inwhich a stem cell matures is critical to the differentiated cells thatresult from the maturation process. Embodiments of the invention arefurther based on the discovery that pancreatic endocrine precursor cellsmay find particular utility as a therapeutic for treatment of Type 2diabetes (T2D) in a subject, wherein the subject is a candidate fortreatment with one or more small molecule anti-diabetic drugs.Embodiments of the invention are further based on the fortuitous findingthat treatment with a combination of pancreatic endocrine precursorcells and a small molecule anti-diabetic drug is useful in the treatmentof T2D, obesity, glucose intolerance and/or insulin resistance.Alternatively, the methods described herein may be used to improveglycemic control. Additional embodiments of the invention are furtherbased on the fortuitous finding that the transplantation of pancreaticendocrine precursor cells alone produce weight loss in a subject.Furthermore, the weight loss was also achieved by transplanting moredifferentiated cells (i.e. Stage 5, Stage 6 or Stage 7 cells).

In a first embodiment, there is provided a method for treating Type 2diabetes (T2D) in a subject, the method including: (a) implanting apopulation of pancreatic endocrine progenitor cells into the subject;and (b) maturing in vivo the population of pancreatic endocrineprogenitor cells to produce a population including pancreatic endocrinecells.

In a further embodiment, there is provided a method for treating Type 2diabetes (T2D) in a subject, the method including: implanting apopulation of pancreatic endocrine progenitor cells into the subject.

In a further embodiment, there is provided a method for treating Type 2diabetes (T2D) in a subject, the method including: (a) implanting apopulation of pancreatic endocrine progenitor cells into the subject,wherein the subject is a candidate for treatment with one or more smallmolecule anti-diabetic drugs; and (b) maturing in vivo the population ofpancreatic endocrine progenitor cells to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a method for treating Type 2diabetes (T2D) in a subject, the method including: implanting apopulation of pancreatic endocrine progenitor cells into the subject,wherein the subject is a candidate for treatment with one or more smallmolecule anti-diabetic drugs.

In a further embodiment, there is provided a method for treating Type 2diabetes (T2D) in a subject, the method including: (a) implanting apopulation of pancreatic endocrine progenitor cells into the subject,wherein the subject is being treated with one or more small moleculeanti-diabetic drugs; and (b) maturing in vivo the population ofpancreatic endocrine progenitor cells to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a method for treating Type 2diabetes (T2D) in a subject, the method including: implanting apopulation of pancreatic endocrine progenitor cells into the subject,wherein the subject is being treated with one or more small moleculeanti-diabetic drugs.

In a further embodiment, there is provided a method for treating obesityin a subject, the method including: (a) implanting a population ofpancreatic endocrine progenitor cells into the subject; and (b) maturingin vivo the population of pancreatic endocrine progenitor cells toproduce a population including pancreatic endocrine cells.

In a further embodiment, there is provided a method for treating obesityin a subject, the method including: implanting a population ofpancreatic endocrine progenitor cells into the subject.

In a further embodiment, there is provided a method for treating controlweight gain in a subject, the method including: (a) implanting apopulation of pancreatic endocrine progenitor cells into the subject;and (b) maturing in vivo the population of pancreatic endocrineprogenitor cells to produce a population including pancreatic endocrinecells.

In a further embodiment, there is provided a method for treating controlweight gain in a subject, the method including: implanting a populationof pancreatic endocrine progenitor cells into the subject.

In a further embodiment, there is provided a method for treating obesityin a subject, the method including: (a) administering a therapeuticallyeffective amount of one or more small molecule anti-diabetic drugs tothe subject; (b) implanting a population of pancreatic endocrineprogenitor cells into the subject; and (c) maturing in vivo thepopulation of pancreatic endocrine progenitor cells to produce apopulation including pancreatic endocrine cells.

In a further embodiment, there is provided a method for treating obesityin a subject, the method including: (a) administering a therapeuticallyeffective amount of one or more small molecule anti-diabetic drugs tothe subject; and (b) implanting a population of pancreatic endocrineprogenitor cells into the subject.

In a further embodiment, there is provided a method for treating controlweight gain in a subject, the method including: (a) administering atherapeutically effective amount of one or more small moleculeanti-diabetic drugs to the subject; (b) implanting a population ofpancreatic endocrine progenitor cells into the subject; and (c) maturingin vivo the population of pancreatic endocrine progenitor cells toproduce a population including pancreatic endocrine cells.

In a further embodiment, there is provided a method for treating controlweight gain in a subject, the method including: (a) administering atherapeutically effective amount of one or more small moleculeanti-diabetic drugs to the subject; and (b) implanting a population ofpancreatic endocrine progenitor cells into the subject.

In a further embodiment, there is provided a method for treatingobesity, glucose intolerance or insulin resistance in a subject withT2D, the method including: (a) implanting a population of pancreaticendocrine progenitor cells into the subject; and (b) maturing in vivothe population of pancreatic endocrine progenitor cells to produce apopulation including pancreatic endocrine cells.

In a further embodiment, there is provided a method for treatingobesity, glucose intolerance or insulin resistance in a subject withT2D, the method including: implanting a population of pancreaticendocrine progenitor cells into the subject.

In a further embodiment, there is provided a method for treatingobesity, glucose intolerance or insulin resistance in a subject withT2D, the method including: (a) administering a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs to the subject;(b) implanting a population of pancreatic endocrine progenitor cellsinto the subject; and (c) maturing in vivo the population of pancreaticendocrine progenitor cells to produce a population including pancreaticendocrine cells.

In a further embodiment, there is provided a method for treatingobesity, glucose intolerance or insulin resistance in a subject withT2D, the method including: (a) administering a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs to the subject;and (b) implanting a population of pancreatic endocrine progenitor cellsinto the subject.

In a further embodiment, there is provided a method improving glycemiccontrol in a subject with T2D, the method including: (a) implanting apopulation of pancreatic endocrine progenitor cells into the subject,wherein the subject is a candidate for treatment with one or more smallmolecule anti-diabetic drugs; and (b) maturing in vivo the population ofpancreatic endocrine progenitor cells to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a method improving glycemiccontrol in a subject with T2D, the method including: implanting apopulation of pancreatic endocrine progenitor cells into the subject,wherein the subject is a candidate for treatment with one or more smallmolecule anti-diabetic drugs.

In a further embodiment, there is provided a method improving glycemiccontrol in a subject with T2D, the method including: (a) administering atherapeutically effective amount of one or more small moleculeanti-diabetic drugs to the subject; (b) implanting a population ofpancreatic endocrine progenitor cells into the subject, wherein thesubject is a candidate for treatment with one or more small moleculeanti-diabetic drugs; and (c) maturing in vivo the population ofpancreatic endocrine progenitor cells to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a method improving glycemiccontrol in a subject with T2D, the method including: (a) administering atherapeutically effective amount of one or more small moleculeanti-diabetic drugs to the subject; and (b) implanting a population ofpancreatic endocrine progenitor cells into the subject, wherein thesubject is a candidate for treatment with one or more small moleculeanti-diabetic drugs.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating Type 2 diabetes (T2D)in a subject, wherein the cells are suitable for implanting into thesubject; and maturing in vivo to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating Type 2 diabetes (T2D)in a subject, wherein the cells are suitable for implanting into thesubject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and one or more small moleculeanti-diabetic drugs for treating Type 2 diabetes (T2D) in a subject,wherein the cells are suitable for implanting into the subject; andmaturing in vivo to produce a population including pancreatic endocrinecells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and one or more small moleculeanti-diabetic drugs for treating Type 2 diabetes (T2D) in a subject,wherein the cells are suitable for implanting into the subject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating obesity in a subject,wherein the cells are suitable for implanting into the subject; andmaturing in vivo to produce a population including pancreatic endocrinecells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating obesity in a subject,wherein the cells are suitable for implanting into the subject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating control weight gainin a subject, wherein the cells are suitable for implanting into thesubject; and maturing in vivo to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating control weight gainin a subject, wherein the cells are suitable for implanting into thesubject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for treatingobesity in a subject, wherein the cells are suitable for implanting intothe subject and maturing in vivo to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for treatingobesity in a subject, wherein the cells are suitable for implanting intothe subject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for treatingcontrol weight gain in a subject, wherein the cells are suitable forimplanting into the subject and maturing in vivo to produce a populationincluding pancreatic endocrine cells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for treatingcontrol weight gain in a subject, wherein the cells are suitable forimplanting into the subject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating obesity, glucoseintolerance or insulin resistance in a subject with T2D, wherein thecells are suitable for implanting into the subject and maturing in vivoto produce a population including pancreatic endocrine cells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for treating obesity, glucoseintolerance or insulin resistance in a subject with T2D, wherein thecells are suitable for implanting into the subject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for treatingobesity, glucose intolerance or insulin resistance in a subject withT2D, wherein the cells are suitable for implanting into the subject andmaturing in vivo to produce a population including pancreatic endocrinecells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for treatingobesity, glucose intolerance or insulin resistance in a subject withT2D, wherein the cells are suitable for implanting into the subject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for improving glycemic control ina subject with T2D, wherein the cells are suitable for implanting intothe subject and maturing in vivo to produce a population includingpancreatic endocrine cells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells for improving glycemic control ina subject with T2D, wherein the cells are suitable for implanting intothe subject.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for improvingglycemic control in a subject with T2D, wherein the cells are suitablefor implanting into the subject and maturing in vivo to produce apopulation including pancreatic endocrine cells.

In a further embodiment, there is provided a use of a population ofpancreatic endocrine progenitor cells and a therapeutically effectiveamount of one or more small molecule anti-diabetic drugs for improvingglycemic control in a subject with T2D, wherein the cells are suitablefor implanting into the subject.

In a further embodiment, there is provided a commercial packageincluding: (a) a population of pancreatic endocrine progenitor cells;and (b) a therapeutically effective amount of one or more small moleculeanti-diabetic drugs.

In a further embodiment, there is provided a commercial packageincluding: (a) a population of pancreatic endocrine progenitor cells;and (b) a therapeutically effective amount of one or more small moleculeanti-diabetic drugs.

The method may further include treating the subject with one or moresmall molecule anti-diabetic drugs. The one or more small moleculeanti-diabetic drugs may be selected from the following: ofdipeptidyl-peptidase 4 (DPP-4) inhibitors; thiazolidinediones; andbiguanides. The anti-diabetic drug may be selected from the groupincluding of: sitagliptin; metformin; and rosiglitazone. The one or moresmall molecule anti-diabetic drugs may be selected from the following:meglitinides; sulfonylureas; dipeptidyl-peptidase 4 (DPP-4) inhibitors;biguanides; thiazolidinediones; alpha-glucosidase inhibitors;sodium-glucose transporter 2 (SGLT-2) inhibitors; and bile acidsequestrants. The small molecule anti-diabetic drug may be selected fromthe group including of: repaglinide; nateglinide; glipizide;glimepiride; glyburide; saxagliptin; sitagliptin; linagliptin;metformin; rosiglitazone; pioglitazone; acarbose; miglitol;canagliflozin; dapagliflozin; empagliflozin; and colsevelam. Theanti-diabetic drug may be sitagliptin. The anti-diabetic drug may bemetformin. The anti-diabetic drug may be rosiglitazone.

The pancreatic endocrine progenitor cells may mature in vivo to producea population comprising at least 2% pancreatic endocrine cells. Thepancreatic endocrine progenitor cells may be encapsulated. Thepancreatic endocrine progenitor cells may be unencapsulated. Thepancreatic endocrine progenitor cells may be macro-encapsulated. Thepancreatic endocrine progenitor cells may be micro-encapsulated. Thepopulation including pancreatic endocrine cells may be a mixedpopulation. The population including pancreatic endocrine cells mayinclude mature islet cells. The population including pancreaticendocrine cells may include mature pancreatic endocrine cells. Thepopulation including pancreatic endocrine cells may include beta-cells.The population including pancreatic endocrine cells may includealpha-cells.

The commercial package may further include instructions for thetreatment of T2D. The commercial package may further includeinstructions for the treatment of obesity. The commercial package mayfurther include instructions for glycemic control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a graph depicting the fasting body weights of the micefrom Example 1 at the specified days following administration of a lowfat diet (10% Fat), diets high in fat (45% Fat; 60% Fat), or a high fat,high carbohydrate diet (Western).

FIG. 1B shows a graph depicting the fasting blood glucose levels of themice from Example 1 at the specified days following administration of alow fat diet (10% Fat), diets high in fat (45% Fat; 60% Fat), or a highfat, high carbohydrate diet (Western).

FIG. 1C shows a graph of the raw values and area under the curve of theblood glucose values during an oral glucose challenge of the mice inExample 1 mice at Day 47

FIG. 1D shows a graph of the insulin levels of the Example 1 mice insamples collected during the oral glucose challenge at Day 47.

FIG. 1E shows a graph of the results of an insulin tolerance testperformed at Day 42 on the mice of Example 1.

FIG. 1F shows the results of dual-energy X-ray absorptiometry (DEXA)assessment of recent fat of a subset of the mice of Example 1 at Day 43.

FIG. 1G shows the leptin levels measured at days 47 through 49 and aftera four to six hour morning fast of the mice of Example 1.

FIG. 2A shows a graph of human C-peptide levels measured after anovernight fast and 40 minutes after an oral mixed-meal challenge at 8,12, 16, and 20 weeks after transplant in the mice of Example 2.

FIGS. 2B and 2C show the results of human C-peptide measurement at 18weeks post-transplant following an intraperitoneal glucose tolerancetest (ipGTT) for mice of Example 2 wherein FIG. 2B are the normalizedbaseline levels and FIG. 2C are the raw levels (ng/mL).

FIGS. 2D-2F show the results of human insulin and glucagon at 24 weekspost-transplant following an arginine tolerance test in the mice ofExample 3, wherein (E) shows glucagon levels at 0 and 15 minutes intransplant recipients, and (F) shows glucagon levels in sham-treatedmice (Sham, striped bars) and transplant recipients (Tx, solid bars) at15 minutes only.

FIG. 3 shows a graph with the percentage of cells with devices that wereimmuno-reactive for insulin, glucagon, or both hormones (Ins+/Gcg+).

FIGS. 4A and 4B show graphs depicting the HbA1C levels in the mice ofExample 4 measured at 12 and 24 weeks post-transplant.

FIG. 4C shows a graph depicting blood glucose levels 20 weekspost-transplant and measured after an overnight fast and 40 minutesfollowing an oral meal challenge in the mice of Example 4.

FIGS. 4D and 4E show graphs depicting the results of intraperitonealglucose tolerance testing performed 18 weeks (4D) and 24 weeks (4E)post-transplant in the mice of Example 4.

FIG. 4F shows a graph depicting the results of insulin tolerance testingperformed 22 weeks post-transplant in the mice of Example 4.

FIGS. 5A-5F show diet-induced obesity was reversed following progenitorcell transplantation combined with an antidiabetic Drug, wherein fastingbody weight was assessed in mice fed a 10% fat diet without drug(black/gray; all panels; n=8 mice), 60% fat diet without drug (A and B;n=7-8 mice per group), 60% fat diet plus metformin (A and C; n=7-8 miceper group), 60% fat diet plus sitagliptin (A and D; n=8 mice per group),or 60% fat diet plus rosiglitazone (A and E; n=8 mice per group). Bodyweight tracking for sham mice from all treatment groups is shown in (A).Sham mice (solid lines, closed symbols) and transplant recipients (Tx;dashed lines, open symbols) from each treatment group are shown togetherwith the LFD control as a reference (B-E). The change in body weightfrom day 2 to day 12 is shown in box-and-whisker plots to the right ofeach line graph, with each data point representing an individual mouse.Data on line graphs are represented as mean±SEM. (F) Body weightpre-transplant (day 2) and post-transplantation (day 75).

FIG. 5G shows a graph depicting the relative epididymal fat pad weightto body weight of Example 6 mice at 20 weeks post transplant.

FIG. 5H shows a graph of plasma mouse leptin levels in Example 6 miceassessed 20 weeks post-transplant.

FIGS. 6A-6E show graphs depicting the results of oral glucose tolerancetesting performed on Example 6 mice 12 weeks post-transplant.

FIG. 6F shows a graph depicting mouse C-peptide levels for Example 6mice 4 weeks (6F) post-transplant either before (o) or after 60 minutesafter (60) a intraperitoneal injection of glucose.

FIG. 6G shows the results of human C-peptide secretion in the Example 6mice measured after an overnight fast and 60 minutes following anintraperitoneal glucose challenge.

FIGS. 7A and 7C show graphs of the results of oral glucose tolerancetests at day 5 (7A) and day 32 (7C) after administration of the diets ofExample 1 to the mice (these are additional time points that go withFIG. 1).

FIGS. 7B and 7D show graphs of the body weight of the Example 1 mice atday 5 (7B) and day 32 (7D).

FIGS. 7E, 7F, and 7G show plasma levels, following a four to six hourmorning fast, in the Example 1 mice between days 47 and 49. 7E is agraph of free fatty acids, 7F is a graph of triglycerides and 7G is agraph of cholesterol.

FIG. 8 shows 4 graphs of flow cytometry results of key markers of theStage 4, day 4 cells of Example 2 showing co-expression of synaptophysinand NKX6.1, chromogranin and NKX2.2, PDX1 and Ki67, and PAX4 and OCT3/4.

FIGS. 9A and 9B show graphs of the results of intraperitoneal glucosetolerance tests performed on the mice of Example 4 at 18 weeks (9A) and24 weeks (9B) post-transplant.

FIGS. 9C and 9D show graphs of the results of insulin tolerance testsperformed on the mice of Example 4 at 22 weeks post-transplant.

FIGS. 10A, 10B and 10C show graphs of the beta cell mass (10A), alphacell mass (10B) and the ratio of insulin to glucagon (10C)immuno-staining in pancreas sections from mice of Example 4, 29 weekspost-transplantation or sham surgery and at 36 weeks afteradministration of the diets.

FIGS. 10D-10G show graphs of the body weight (10D), epididymal fatweight (10E), plasma leptin levels (10F) and liver weight (10G) of themice of Example 4.

FIG. 11A shows 2 graphs of fasting blood glucose (left) and body weightlevels (right) of a subset of the Example 6 mice.

FIG. 11B shows 2 graphs of fasting blood glucose (left) and body weightlevels (right) following administration of either a low fat diet or ahigh fat diet to the Example 6 mice.

FIG. 11C shows a graph of the results of an oral glucose tolerance test2 weeks after administration of either a low fat diet or a high fat dietto Example 6 mice.

FIG. 11D shows a graph of the results of an insulin tolerance test 3weeks after administration of either a low fat diet or a high fat dietto Example 6 mice.

FIGS. 12A and 12B show two graphs of body weight in grams plottedagainst time post transplant in days for female (12A) and male (12B)mice, wherein 1 M=1 million, 5 M=5 million, S4=Stage 4 cells andS7=Stage 7 cells. Mice were weighted about 4 days before transplant andabout every 2-4 weeks after the transplant following a 4 hour fast(usually 7 am-11 am) and zero (0) is the day of the transplant.

FIGS. 13A and 13B show two graphs of body weight in grams plottedagainst time post transplant in weeks for female (13A) and male (13B)mice, wherein 1 M=1 million, 5 M=5 million, S4=Stage 4 cells andS7=Stage 7 cells. Mice were weighted at 2, 4, 8, 12, 16, 20 and 24 weekspost-transplant following an overnight fast (usually 5 pm-8 am ˜15 hrs.)and zero (0) is the day of the transplant.

FIGS. 1.4A and 14B show two graphs of body weight in grams (BW—14A) andblood glucose in mM (BG-4 hour fast—14B) comparing transplanted Stage 7cells to human islet transplants in mice and non-diabetic mice, whereina subset of human islet transplant mice were non-diabetic (i.e. not STZtreated) and the remainder were “diabetic” (i.e. STZ treated).

FIGS. 15A-15C show three graphs comparing control mice to micetransplanted with Stage 7 cells (S7) and mice transplanted with humanislet cells (same mice from FIG. 14), where (15A) mouse Leptin (ng/ml)was measured in random fed mice, (15B) fat mass (g) from dual-energyX-ray absorptiometry (DEXA) and (15C) mouse Leptin (ng/ml) in random fedmice divided by grams of fat.

FIGS. 16A-16C show three graphs comparing control mice to micetransplanted with Stage 7 cells (S7) and mice transplanted with humanislet cells (same mice from FIGS. 14 and 15), where (16A) lean bodymass, (16B) fat mass and (16C) % body fat measured by dual-energy X-rayabsorptiometry (DEXA).

FIGS. 17A-17D show four graphs comparing control mice to micetransplanted with Stage 7 cells (S7) and mice transplanted with humanislet cells (same mice from FIGS. 14 and 15) FIG. (17A) is the perirenalfat weight of either the left kidney or right kidney (graft bearingkidney) or both. FIGS. 17B-D are the weights of the epididymal fat,mesenteric fat, and all fat pads combined, respectively.

FIGS. 18A-18C show 3 graphs comparing Stage 4 and Stage 6 celltransplants to control mice (no cell transplant—sham operation) insubcutaneous deviceless and kidney capsule (KC) implanted mice showingC-peptide ng/ml (18A); body weight (18B); and weekly fasting bloodglucose (18C).

FIG. 19 shows a graph tracking of human C-peptide in individual mice(animals numbers on X-axis) at the indicating time points posttransplant of Stage 7 cells within TheraCyte devices, transplantedsubcutaneously (S7+ Tc Tx), or without encapsulation and transplantedbeneath the kidney capsule (KC), with transplants unsuccessful in animalnumbers 10 and 40.

FIG. 20 shows graph tracking of human C-peptide in mice at theindicating time points post transplant of Stage 7 cells within TheraCytedevices (TC), transplanted subcutaneously, or without encapsulation andtransplanted beneath the kidney capsule (KC).

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention will be betterunderstood when read in conjunction with the appended figures. For thepurpose of illustrating the invention, the figures demonstrateembodiments of the present invention. However, the invention is notlimited to the precise arrangements, examples, and instrumentalitiesshown.

The present invention is directed to the discovery that transplantationof pancreatic endocrine precursor cells into a subject for furtherdifferentiation into functional pancreatic endocrine cells, alone or incombination with the administration of a therapeutically effectiveamount of an anti-diabetic drug, results in weight loss in the subject.Additionally, co-therapy that includes transplantation of pancreaticendocrine precursor cells for further differentiation into functionalpancreatic endocrine cells, alone or in combination with theadministration of a therapeutically effective amount of selectedanti-diabetic drugs, results in improvement in glucose tolerance andinsulin resistance in Type 2 diabetes (T2D) model mammals.

Thus, embodiments of the present invention provide a method for treatingobesity in a subject comprising: (a) implanting a population ofpancreatic endocrine progenitor cells into the subject; and (b) maturingin vivo the population of pancreatic endocrine precursor cells.Optionally, the method may include administering a therapeuticallyeffective amount of one or more anti-diabetic drugs to the subject.Alternatively, embodiments of the present invention provide a method fortreating obesity in a subject comprising: (a) implanting a population ofpancreatic endocrine progenitor cells into the subject; and (b) maturingin vivo the population of pancreatic endocrine precursor cells toproduce a population of cells comprising about 2% or more of pancreaticendocrine cells. Optionally, the method may include administering atherapeutically effective amount of one or more anti-diabetic drugs tothe subject. Additionally, embodiments of the invention may provide amethod for treating obesity, glucose intolerance and insulin resistancein a subject with T2D comprising: (a) implanting a population ofpancreatic endocrine progenitor cells into the subject; (b) maturing invivo the population of pancreatic endocrine precursor cells; and (c)administering a therapeutically effective amount of one or moreanti-diabetic drugs to the subject, wherein the anti-diabetic drugs areselected from the group consisting of dipeptidyl-peptidase 4 inhibitors,thiazolidinediones, and biguanides. Alternatively, other embodiments ofthe present invention provide a method for treating obesity in a subjectcomprising: (a) implanting a population of pancreatic endocrineprogenitor cells into the subject; (b) maturing in vivo the populationof pancreatic endocrine precursor cells to produce a population of cellscomprising about 2% or more of pancreatic endocrine cells. Optionally,the method includes administering a therapeutically effective amount ofone or more anti-diabetic drugs to the subject. Additionally, theinvention provides a method for treating obesity, glucose intoleranceand insulin resistance in a subject with T2D comprising: (a) implantinga population of pancreatic endocrine precursor cells into the subject;(b) maturing in vivo the population of pancreatic endocrine precursorcells to produce a population comprising about 2% or more of pancreaticendocrine cells; and (c) administering a therapeutically effectiveamount of one or more anti-diabetic drugs to the subject, wherein theanti-diabetic drugs are selected from the group consisting ofdipeptidyl-peptidase 4 inhibitors, thiazolidinediones, and biguanides.Still in some embodiments, the various indicated pancreaticendoderm-lineage cells or as defined herein as pancreatic progenitorcells, which includes cells of Stages 4, 5, 6 and 7 or pancreaticendocrine cells derived from an in vitro maturation process as describedherein may be used alone or in combination, but independent of anyanti-diabetic drug, and used to treat obesity, glucose intolerance,glycemic control and insulin resistance.

The stem cells or pluripotent stem cells used to provide the pancreaticendocrine precursor cells useful in the invention are undifferentiatedcells defined by their ability, at the single cell level, to bothself-renew and differentiate including but not limited to humanembryonic stem cells, induced pluripotent stem cells, human umbilicalcord tissue-derived cells, human amniotic fluid-derived cells, humanplacental-derived cells, and human parthenote-derived stem cells. Thestem cells are also characterized by their ability to differentiate invitro into functional cells of various cell lineages from multiple germlayers (endoderm, mesoderm, and ectoderm). The stem cells also give riseto tissues of multiple germ layers following transplantation andcontribute substantially to most, if not all, tissues followinginjection into blastocysts.

The stem cells are differentiated, which differentiation is the processby which an unspecialized (“uncommitted”) or less specialized cellacquires the features of a specialized cell. A differentiated cell isone that has taken on a more specialized (“committed”) position withinthe lineage of a cell. The term “committed”, when applied to the processof differentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.

Markers may be used to characterize the stem cells and the variousdifferentiated cells. “Markers”, as used herein, are nucleic acid orpolypeptide molecules that are differentially expressed in a cell ofinterest. In this context, differential expression means an increasedlevel for a positive marker and a decreased level for a negative markeras compared to an undifferentiated cell, a cell at another stage ofdifferentiation within the same lineage, or a cell of a differentlineage. The detectable level of the marker nucleic acid or polypeptideis sufficiently higher or lower in the cells of interest compared toother cells, such that the cell of interest can be identified anddistinguished from other cells using any of a variety of methods knownin the aft.

The differentiation process is often viewed as progressing through anumber of consecutive stages. For purposes of one embodiment of thisinvention, in the step-wise differentiation, “Stage 1” refers to thefirst step in the differentiation process in which pluripotent stemcells are differentiated into cells expressing markers characteristic ofthe definitive endoderm (“Stage 1 cells”). “Stage 2” refers to thesecond step, the differentiation of cells expressing markerscharacteristic of the definitive endoderm cells into cells expressingmarkers characteristic of gut tube cells (“Stage 2 cells”). “Stage 3”refers to the third step, differentiation of cells expressing markerscharacteristic of gut tube cells into cells expressing markerscharacteristic of foregut endoderm cells (“Stage 3 cells”). “Stage 4”refers to the fourth step, the differentiation of cells expressingmarkers characteristic of foregut endoderm cells into cells expressingmarkers characteristic of pancreatic endocrine precursor cells (“Stage 4cells”). In other embodiments, the step-wise differentiation processincludes differentiating pancreatic foregut precursor cells intopancreatic endocrine precursor cells (“Stage 5 cells”) or immatureendocrine cells (“Stage 6 cells”) and, subsequently, furtherdifferentiation into more mature endocrine cells (“Stage 7 cells”), eachstage identified by specific markers characteristic of the cells at thegiven stage. The actual numbered stage, however, is not limiting as aparticular population of cells is defined by any of cell types in thepopulation, which cell types are identified by the markers they expressrelative to other cell types in the same population.

It is to be noted that not all cells in a particular population progressthrough the stages at the same rate. Consequently, it is not uncommon inin vitro cell cultures to detect the presence of cells that haveprogressed less, or more, down the differentiation pathways than themajority of cells present in the population, particularly at the laterdifferentiation stages. For purposes of illustrating the presentinvention, characteristics of the various cell types associated with theabove-identified stages are described herein.

“Definitive endoderm” or “endoderm-lineage cells” or equivalents thereofas used herein, refers to cells that express at least one of thefollowing markers: FOXA2 (also known as hepatocyte nuclear factor 3-beta(“HNF3-beta”)), GATA4, SOX17, CXCR4, Brachyury, Cerberus, OTX2,goosecoid, C-Kit, CD99, and MIXL1. Markers characteristic of thedefinitive endoderm cells are CXCR4, FOXA2, and SOX17.

“Gut tube cells” or equivalents thereof, as used herein, refers to cellsderived from definitive endoderm that may be characterized by theirsubstantially increased expression of HNF4-alpha over that expressed bydefinitive endoderm cells.

“Foregut endoderm cells” or “PDX1 pancreatic endoderm cells” orequivalents thereof, as used herein, refers to cells that express atleast one of the following markers: PDX1, FOXA2, CDX2, SOX2, andHNF4-alpha. Foregut endoderm cells may be characterized by an increasein expression of PDX1 compared to gut tube cells.

“Pancreatic foregut precursor cells”, or “pancreatic progenitor cells”or “Stage 4 cells” (S4 cells), or equivalents thereof as used herein,refers to cells that express at least one of the following markers:PDX1, NKX6.1, HNF6, SOX9, FOXA2, PTF1a, PROX1 and HNF4 alpha. Morespecifically, pancreatic foregut precursor cells may be identified bybeing positive for the expression of at least one of PDX1, NKX6.1 andSOX9 and with low expression of NGN3 and NeuroD.

“Pancreatic endocrine precursor cells” or “Stage 5 cells” (Stage 5cells) or “pancreatic endoderm cells” or equivalents thereof, as usedherein, refers to pancreatic endoderm cells capable of becoming apancreatic hormone expressing cell and that express at least one of thefollowing markers: NGN3; NKX2.2; NeuroD1; ISL1; PDX1; PAX4; PAX6;NKX6.1, or ARX. Pancreatic endocrine precursor cells may becharacterized by their expression of NKX2.2, NKX6.1, PDX1 and NeuroD1.

“Pancreatic endocrine cells,” or “Pancreatic hormone expressing cell”,or “Cells expressing markers characteristic of the pancreatic endocrinelineage” or “Stage 6 or 7 cells” (S6 or S7 cells) or equivalents thereofas used herein, refer to cells capable of expressing at least one of thefollowing hormones: insulin, glucagon, somatostatin, ghrelin, andpancreatic polypeptide. In addition to these hormones, markerscharacteristic of pancreatic endocrine cells include one or more ofNGN3, NeuroD1, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, MNX1 (Hb9) andPAX6. Pancreatic endocrine cells expressing markers characteristic ofbeta-cells can be characterized by their expression of insulin and atleast one of the following transcription factors: PDX1, NKX2.2, NKX6.1,NeuroD1, ISL1, HNF3-beta, MAFA, MNX1 and PAX6. “More mature endocrinecells” express markers characteristic of pancreatic endocrine cells, buthave a more mature phenotype as compared to immature endocrine cellsmeaning that the more mature endocrine cells not only are insulin+,MAFA+, NKX6.1+ and PDX1+, but also display glucose responsive insulinsecretion.

“Functional pancreatic beta-cell” or “beta-cell” equivalents thereof asused herein, refer to an insulin positive cell capable of being glucoseresponsive and positive for PDX-1 and NKX6.1 as referred to inUS20150353895; or a “SC-beta-cell” as referred to in WO2015002724. Stillin some embodiments, the functional pancreatic beta cell expresses atleast one marker characteristic of an endogenous mature pancreaticbeta-cell selected from the group consisting of insulin, C-peptide, PDX1, MAFA, NKX6-1, PAX6, NEUROD1 (or NEUROD), glucokinase (GCK), SLC2A 1,PCS1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2, PAX4,IRX1, and IRX2.

“Appropriate growth factors” or “appropriate factors” or equivalentsthereof refers to those particular growth factors and agents used todifferentiate a population of cells from one stage to another or furtherdifferentiated stage. The appropriate factors or agents for each of thedifferentiation steps or stages are described in detail in D'Amour, K A.et al. 2005, D'Amour, K A. et al. 2006, Kroon E. et al. 2008, Schulz T.et al. 2012, Rezania A. et al. 2014, Bruin J. et al. 2014, Pagliuca F W.et al. 2014, Agulnick A. D. et al. 2015 (see also WO/2014/160413) andthe like.

Cells useful in the methods of the invention may be any population ofStage 4 or Stage 5 pancreatic endocrine precursor cells, or Stage 6 orStage 7 pancreatic endocrine cells and all cells up to, but notincluding mature beta-cells, and are collectively referred to as“pancreatic endocrine progenitor cells”. Alternatively, cells useful inthe methods may further include endocrine cells and in vitro maturedpancreatic endocrine cells. Alternatively, cells useful in the inventionmay be any of a population of immature pancreatic endocrine cells ormore mature endocrine cells that are not only insulin+, MAFA+, NKX6.1+,and PDX1+ but also display glucose responsive insulin secretion.Preferably, the cells used in the invention are pancreatic endocrineprecursor cells.

“Subject” or equivalents thereof as used herein refers to an animal,preferably a mammal, most preferably a human adult or child. “Obesity”as used herein means an accumulation of body fat that is undesirable orequal to or greater than about 20% of a subject's ideal body weight.“Effective amount” or equivalents thereof of a compound, growth factoror agent refers to that concentration of the compound, growth factor oragent that is sufficient in the presence of the remaining components ofthe cell culture medium to either maintain the cell in anundifferentiated state (e.g. pluripotent cells) or promotedifferentiation of a cell. This concentration is readily determined byone of ordinary skill in the art and for many of the cell typesdescribed herein, the effective amount is described in detail in atleast D'Amour, K A. et al. 2005, D'Amour, K A. et al. 2006, Kroon E. etal. 2008, Schulz T. et al. 2012, Rezania A. et al. 2014, Bruin J. et al.2014, Pagliuca F W. et al. 2014, Agulnick A. D. et al. 2015 and thelike. In some embodiments, the therapeutic effective amount is ascompared to cell cultures which do not receive the same treatment ortherapeutic effective amount of the compound, growth factor or agent.“Therapeutic effective amount” or equivalents thereof as used hereinrefer to one or more small molecule anti-diabetic drugs given alone orin combination to provide the desired benefit to the subject.

In some embodiments, the methods and co-therapies of the inventionutilize anti-diabetic drugs in addition to implanted, differentiatedcells, to treat one or both of obesity and glucose intolerance in asubject. By “anti-diabetic drug” or “anti-diabetic medication” is meanta medication, agent or the like that acts to lower blood sugar levels ina person with T2D.

The anti-diabetic drugs useful in the invention may work by any of anumber of ways to lower blood sugar, including simulating insulinrelease and production from the pancreas, inhibiting glucose releasefrom the liver, inhibiting stomach enzymes that break-downcarbohydrates, improving cells sensitivity to insulin, inhibitingglucose reabsorption in the kidneys, or slowing food motility in thestomach. These anti-diabetic drugs include the following oralmedications: meglitinides, for example such as repaglinide andnateglinide; sulfonylureas, such as glipizide, glimepiride, andglyburide; dipeptidyl-peptidase 4 (“DPP-4”) inhibitors such assaxagliptin, sitagliptin, and linagliptin; biguanides such as metformin;thiazolidinediones such as rosiglitazone and pioglitazone;alpha-glucosidase inhibitors such as acarbose and miglitol;sodium-glucose transporter 2 (“SGLT2”) inhibitors such as canagliflozin,dapagliflozin, and empagliflozin; and bile acid sequestrants such ascolsevelam. Alternatively, injectable medications, such as amylinmimetics, including pranlintide, and incretin mimetics, including GLP-1receptor agonists, such as exenatide and liraglutide.

The anti-diabetic drugs are used in “therapeutically effective amounts”,meaning the amount of anti-diabetic drug that elicits the biological ormedicinal response in a tissue system, animal or human that is beingsought by a researcher, veterinarian, medical doctor, or otherclinician, which includes alleviation of one or more of the symptoms ofthe disease or disorder being treated or the reduction of the severityof one or more symptom of the disease or disorder being treated.

Any pluripotent stem cells may be used in the invention to provide thepancreatic endocrine precursor, pancreatic foregut precursor, and matureendocrine cells. Exemplary types of pluripotent stem cells that may beused include established lines of pluripotent cells, includingpre-embryonic tissue (such as, a blastocyst), embryonic tissue, or fetaltissue taken any time during gestation, typically but not necessarily,before approximately io to 12 weeks gestation. Non-limiting examples areestablished lines of human embryonic stem cells (hESCs) or humanembryonic germ cells, such as, any of the current 362 human embryonicstem cell lines listed on the NIH Human Embryonic Stem Cell Registryincluding but not limited to H1 (NIH Code: WA01), H7 (NIH Code: WA07),H9 (NIH Code: WA09) (WiCell Research Institute™, Madison, Wis., USA),SA002 (Cellartis AB Corporation™, Goteburg, Sweden), CyT49 (ViaCyte,Inc.). Pluripotent stem cell markers include, for example, theexpression of one or more of the following: ABCG2, cripto, FOXD3,CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3,SSEA-4, TRA-1-60, TRA-1-81. These may be detectable by RT-PCR or flowcytometry or similar technologies now or later developed.

Cells taken from a pluripotent stem cell population already cultured inthe absence of feeder cells are also suitable. Induced pluripotent cells(IPS), or reprogrammed pluripotent cells, derived from adult somaticcells using forced expression of a number of pluripotent relatedtranscription factors, such as OCT4, NANOG, SOX2, KLF4, and ZFP42 (Loh,Y H. et al. 2011, f; see also IPS, Takahashi, K. and Yamanaka, S. 2006)may also be used. The human embryonic stem cells used in the methods ofthe invention may also be prepared as described by Thomson et al. (U.S.Pat. No. 5,843,780; Thomson, J A. et al. 1998; Thomson, J A. andMarshall, V S. 1998; Thomson, J A. et al. 1995). Mutant human embryonicstem cell lines, such as, BG01v (BresaGen™, Athens, Ga.), or cellsderived from adult human somatic cells, such as, cells disclosed inTakahashi et al. 2007 may also be used. In certain embodiments,pluripotent stem cells suitable for use in the present invention may bederived according to the methods described in: Li et al. 2009; Maheraliet al. 2007; Stadtfeld et al. 2008; Nakagawa et al. 2008; Takahashi etal. 2007; and U.S. Patent App. Pub. No. 2011/0104805. In certainembodiments, pluripotent stem cells suitable for use in the presentinvention may be considered “naïve” and derived according to the methodsdescribed in: Gafni et al. 2013, and Ware et al. 2014.

In certain embodiments, the pluripotent stem cells may be ofnon-embryonic origins. Yet other sources of suitable cells include humanumbilical cord tissue-derived cells, human amniotic fluid-derived cells,human placental-derived cells, and human parthenotes. In one embodiment,the umbilical cord tissue-derived cells may be obtained by the method ofU.S. Pat. No. 7,510,873. In another embodiment, the placentaltissue-derived cells may be obtained using the methods of U.S. PatentApplication Publication No. 2005/0058631. In another embodiment, theamniotic fluid-derived cells may be obtained using the methods of U.S.Patent App. Pub. No. 2007/0122903. Each of these patent applications isincorporated in its entirety herein as it pertains to the isolation andcharacterization of the cells.

Pluripotent stem cells are typically cultured on a layer of feeder cellsthat support the pluripotent stem cells in various ways. Alternatively,the pluripotent stem cells may be cultured in a culture system that isessentially free of feeder cells, but nonetheless supports proliferationof pluripotent stem cells without undergoing substantialdifferentiation. The growth of pluripotent stem cells in feeder-freeculture without differentiation is often supported using a mediumconditioned by culturing previously with another cell type.Alternatively, the growth of pluripotent stem cells in feeder-freeculture without differentiation can be supported using a chemicallydefined medium.

Pluripotent cells may be readily expanded in culture using variousfeeder layers or by using matrix protein coated vessels. Alternatively,chemically defined surfaces in combination with defined media such asmedia sold under the trademark mTeSR™-1 and TeSR™-2 (StemCellTechnologies, Inc.™, Vancouver, B.C., Canada) may be used for routineexpansion of the undifferentiated cells. Pluripotent cells may bereadily removed from culture plates using enzymatic digestion,mechanical separation, or various calcium chelators such asethylenediaminetetraacetic acid (“EDTA”). Alternatively, pluripotentcells may be expanded in suspension in the absence of any matrixproteins or feeder layer.

The pluripotent stem cells may be plated onto a suitable culturesubstrate. An exemplary suitable culture substrate is an extracellularmatrix component, such as those derived from basement membrane or thatmay form part of adhesion molecule receptor-ligand couplings. A suitableculture substrate is a reconstituted basement membrane sold under thetrademark MATRIGEL™ (Corning Incorporated™, Corning, N.Y.).

Other extracellular matrix components and component mixtures known inthe art are suitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparin sulfate, and the like, alone or in variouscombinations.

The pluripotent stem cells may be plated onto the substrate in asuitable distribution and in the presence of a medium, which promotescell survival, propagation, and retention of the desirablecharacteristics.

As pluripotent cells differentiate towards beta-cells, theydifferentiate through various stages each of which may be characterizedby the presence or absence of particular markers. Differentiation of thecells into these stages is achieved by the specific culturing conditionsincluding the presence and lack of certain factors added to the culturemedia. In general, this process involves differentiation of pluripotentstem cells into definitive endoderm cells. These definitive endodermcells may then be further differentiated into gut tube cells, which inturn may then be differentiated into foregut endoderm cells. In oneembodiment, foregut endoderm cells may be differentiated into pancreaticforegut precursor cells which may then be further differentiated intopancreatic endocrine precursor cells. These cells may be yet furtherdifferentiated into pancreatic hormone producing or secreting cells. Inanother embodiment, the foregut endoderm cells may be differentiatedinto pancreatic endocrine precursor cells and further differentiatedinto pancreatic hormone producing or secreting cells.

In certain embodiments of the invention, to arrive at the cellsexpressing markers characteristic of the pancreatic endocrine precursorcells, a protocol starting with pluripotent stem cells is employed. Thisprotocol includes:

Stage 1: Pluripotent stem cells such as embryonic stem cells obtainedfrom cell culture lines are treated with the appropriate factors toinduce formation of definitive endoderm cells.

Stage 2: Cells resulting from Stage 1 are treated with the appropriatefactors to induce formation of cells into markers expressingcharacteristic of gut tube cells.

Stage 3: Cells resulting from Stage 2 cells are treated with theappropriate factors to induce further differentiation into cellsexpressing markers characteristic of foregut endoderm cells.

Stage 4: Cells resulting from Stage 3 are treated with the appropriatefactors to induce further differentiation into cells expressing markerscharacteristic of pancreatic pancreatic endoderm lineage expressing atleast one of the following markers: PDX1, NKX6.1, HNF1 beta, PTF1 alpha,HNF6, HNF4 alpha, SOX9, HB9 or PROX1. Cells expressing markerscharacteristic of the pancreatic endoderm lineage do not substantiallyexpress CDX2 or SOX2.

Stage 5: Cells resulting from Stage 4 are treated with the appropriatefactors to induce further differentiation into cells expressing markerscharacteristic of pancreatic endocrine precursor cells capable ofbecoming a pancreatic hormone expressing cell in particular to maximizeinduction of NGN3. Such a cell can express at least one of the followingmarkers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

Stage 6 or Stage 7: Cells resulting from Stage 5 or 6 are treated withthe appropriate factors to induce further differentiation into cellsexpressing markers characteristic pancreatic endocrine cells expressinginsulin are capable of being glucose responsive and positive for PDX-1and NKX6.1, and often MAFA.

The invention provides methods of treatment, and in particular fortreating subjects suffering from one or more of obesity, glucoseintolerance, and insulin resistance. In one embodiment, the method oftreatment comprises implanting cells obtained or obtainable by a methodof the invention into a subject. In one embodiment, the method oftreatment comprises differentiating pluripotent cells in vitro intopancreatic precursor cells, pancreatic endocrine cells or matureendocrine cells, for example as described herein, and implanting thedifferentiated cells into a subject. In another embodiment, the methodfurther comprises the step of culturing pluripotent stem cells, forexample as described herein, prior to the step of differentiating thepluripotent stem cells. In a still further embodiment, the methodfurther comprises the step of differentiating the cells in vivo afterthe step of implantation. In one embodiment, the subject being treatedby any of the methods is a mammal and preferably is a human.

In one embodiment, the cells may be implanted as dispersed cells orformed into clusters that may be infused into the vascular system, forexample, the hepatic portal vein. Alternatively, the cells may beprovided in a biocompatible, porous, polymeric support, degradabledevices or non-degradable devices, or encapsulated (macro or microencapsulation may be use) to protect the cells from the immune system ofthe host. Cells may be implanted into an appropriate site in a recipientincluding, for example, the liver, muscle adipose, pancreas, renalsubscapular space, omentum, peritoneum, sub-serosal space, intestine,stomach, or a subcutaneous pocket. The site of transplantation, with orwithout a cell receptacle, may be pre-vascularized prior to cellimplantation. For example, a prevascularized subcutaneous site may beprepared for islet cell transplantation (see Pepper AR. et al. 2015). Infact, the inventors have shown that both Stage 4 and Stage 6 cells cansurvive, mature and function (i.e. showing both glucose-induction andC-peptide release) following subcutaneous transplant without a deviceusing the Pepper A R. et al. 2015 methodology (data not shown).

Attempts to shield beta-cells from the immune destruction usingprotective capsules have many challenges (see Tang, Q. and Desai, T A.2016). For example, materials that are used to encapsulate beta-cellsmust be permeable to glucose and insulin while preventing immune cellsand the toxic molecules that they produce from reaching the beta-cells.Sealing beta cells in such capsules can be problematic, since a highproportion of islets die shortly after transplantation as a result ofischemia, a condition that is worsened by encapsulation because thestructure may prevent vascularization of the islets. Moreover,encapsulation can reduce the speed at which beta-cells respond tochanges in blood glucose levels because of the time needed for glucoseand insulin to diffuse across the space between the capsule surface andthe beta-cells. With pancreatic islet clusters, there is one capillaryadjacent to each beta-cell for the efficient coupling of blood glucosechanges with insulin release. Both problems in beta-cell survival andfunction can be exacerbated by an inflammatory foreign-body response(FBR), which is often induced by the material used to encapsulate thecells. Macrophages in the transplant recipients recognize the materialsas foreign and form a fibrous wall to contain them, which may lead tofouling of the device surface and suffocation of the cells within.

To enhance further differentiation, survival or activity of theimplanted cells in vivo, additional factors, such as growth factors,antioxidants, or anti-inflammatory agents may be administered before,simultaneously with, or after administration of the cells. These factorscan be secreted by endogenous cells and exposed to the administeredcells in situ. Implanted cells can be induced to differentiate by anycombination of endogenous and exogenously administered growth factorsknown in the art. Additionally, it may be beneficial to administer oneor more immunosuppressive drugs to the subject pre- or post-cellimplantation to prevent rejection of the implanted cells.

The amount of cells used in implantation depends on a number of factorsincluding the condition of the implantation subject and response to theimplanted therapy and can be determined by one skilled in the art. Thecells can be maintained in vitro on a support prior to implantation intothe patient. Alternatively, the support containing the cells can bedirectly implanted in the patient without additional in vitro culturing.The support can optionally be incorporated with at least onepharmaceutical agent that facilitates the survival and function of thetransplanted cells.

Transplantation of pancreatic progenitor cells to a host for in vivomaturation exposes the cells to many environmental influences, which maybe permissive, prohibitive, detrimental, beneficial or some combinationthereof. Furthermore, the many permutations and combinations of theseinfluences are difficult to predict and the influences are bound to bedependent on many factors (for example, the stage of the cell, the typeof cell, the host environment (including, disease state, medicationsetc.)). For example, cell maturation in a T1D model (i.e. absoluteinsulin deficiency due to islet destruction with a large STZ dose tomimic the autoimmune response in T1D patients, which requires insulinadministration, but depending on the type of insulin treatment, exerciseand food intake may still result in hyperglycemia or hypoglycemia) isconsiderably different in a T2D model (i.e. where there is partialdamage to the islet cells, wherein they still produce insulin, if asomewhat insufficient amount and insulin resistance leads to increasedblood glucose due to inappropriate release of glucose by the liver andinappropriate regulation of metabolism by the CNS). Furthermore, therole of T2D diabetes medications (for example, repaglinide; nateglinide;glipizide; glimepiride; glyburide; saxagliptin; sitagliptin;linagliptin; metformin; rosiglitazone; pioglitazone; acarbose; miglitol;canagliflozin; dapagliflozin; empagliflozin; and colsevelam) onpancreatic progenitor cell maturation in vivo was not previouslyunderstood. In fact Calcineurin inhibitors (for example, Cyclosporineand tacrolimus) are reported to block pancreatic regeneration in aductal ligation model (see for example, Heit, J J. et al. 2006; and Nir,T D. et al. 2007). Furthermore, exposure to a high fat diet did notappear to influence the maturation process in vivo (see Example 3).

It is possible that one might have predicted insulin replacement fromtransplanted beta-cells could be therapeutic in T2D, despite the paucityof evidence to support it. However, one could not predict how thepre-requisite precursor cell maturation period, prior to insulinsecretion, would be impacted when growing within an environment of T2D.There is evidence that both elevated glycemia and insulin resistance(key characteristics of T2D) can impair beta-cell development (see forexample, Jonas, J C. et al. 1999; and Kahraman, S. et al. 2014).Similarly, diet has been shown to influence beta-cell development (seefor example, O'Dowd, J F. and Stocker, C J. 2013). Accordingly, whetheror not the pancreatic precursor cells would mature into beta-cells andsubsequently function appropriately in this environment (hyperglycemia,insulin resistance, similar to T2D), and thereby improve glucosehomeostasis required experimentation.

As reported herein the differentiation of the progenitors is differentin male versus female mice (maturation to glucose-responsive beta-cellsis faster in females than males), something we had not predicted (datanot shown; see also FIGS. 12 and 13) and it has also been discoveredthat the setting of hypothyroidism impairs the differentiation of thepancreatic progenitors (see Bruin, J E. et al. “Hypothyroidism impairshuman stem cell-derived pancreatic progenitor cell maturation in mice”Diabetes (2016) pii: db151439. [Epub ahead of print]). Thusenvironmental influences can indeed disrupt both the maturation andfunction of the transplanted cells, and this may be unpredictable, suchthat one should not have assumed maturation of precursor cells intofunctional beta-cells would occur in the setting of T2D-like parameters.

In terms of the influence of other anti-diabetic medicines, we havepreviously examined short term effect of insulin and exendin-4; bothwere without significant effect, under the conditions tested (see forexample, Bruin, J E. et al. 2013). However, others have reported thatdrugs can impair beta-cell regeneration, such as immunosuppressive drugs(see for example, Nir T. et al. 2007).

Materials and Methods

In Vitro Differentiation of hESCs and Assessment of PancreaticProgenitor Cells

The H1 hESC line was obtained from the WiCell Research Institute™. Allexperiments at The University of British Columbia (UBC) with H1 cellswere approved by the Canadian Stem Cell Oversight Committee and the UBCClinical Research Ethics Board. Pluripotent H1 cells were differentiatedinto pancreatic progenitor cells according to our 14-day, four-stageprotocol as previously described (Bruin et al., 2013). Otherdifferentiation protocols for producing pancreatic progenitors orendrocine precursors and pancreatic endocrine cells are available andhave been shown to develop and mature to become at least insulinsecreting cells in response to physiological glucose levels (Kroon E. etal. 2008; Schulz T. et al. 2012; Rezania et al. 2014; Pagliuca, F W. etal. 2014; Agulnick, A D. et al. 2015; and Russ, H A, et al. 2015)Expression of key pancreatic progenitor cell markers or endocrine cellmarkers are assessed prior to transplantation using well knownmethodologies including custom Taqman™ qPCR Arrays (AppliedBiosystems™).

Flow Cytometry

Differentiated cells were released into a single-cell suspension, fixed,permeabilized, and stained for various intracellular markers, asdescribed previously (Rezania et al., 2012). Dead cells were excludedduring FACS analysis and gating was determined using isotype antibodies.Refer to TABLE 1 for antibody details.

TABLE 1 Antibody information for FACS Type Antibody Source DilutionUnconjugated Primary antibodies Mouse anti-NKX6.1 Developmental StudiesHybridoma Bank  1:400 University of Iowa (Cat #F55A12) Rabbitanti-Synaptophysin Abcam ™ (Cat #ab52636)  1:800 Rabbitanti-Chromogranin A DAKO ™ (Cat# IS502) 1:10 Mouse anti-NKX2.2Developmental Studies Hybridoma Bank  1:100 University of Iowa (Cat#74.5A5) Conjugated primary antibodies Alexa Fluor 647 mouse anti- BD ™cat# 561126 1:10 human Ki67 PE mouse anti-PDX1 BD ™ cat# 562161 1:40 PEmouse anti-human Pax6 BD ™ cat# 561552 1:20 Alexa Fluor 647 mouse anti-BD ™ cat# 560329 1:20 Oct3/4 Secondary antibodies Goat Anti Mouse IgGAF647 Invitrogen ™ (Cat# A21235)  1:4000 PE-Goat anti-Rabbit Fab2 IgGInvitrogen ™ (Cat# A10542)  1:800 (H + L) Isotype control antibodiesPurified Rabbit IgG, k Isotype BD ™ cat# 550875  1:1000 Purified MouseIgG, k Isotype BD ™ cat# 557273 1:50 PE Mouse IgG1, k, Isotype ControlBD ™ cat # 555749 1:40 Alexa Fluor 647 IgG1, Isotype Control BD ™ cat#557732 1:40

Animals

Male SCID-beige mice (C.B-Igh-1b/GbmsTac-Prkdcscid-LystbgN7, 8-10 weeksold; Taconic™) were maintained on a 12 hr light/dark cycle throughoutthe study. All experiments were approved by the UBC Animal CareCommittee and carried out in accordance with the Canadian Council onAnimal Care guidelines.

Diets and Drug Administration

All mice were given ad libitum access to a standard irradiated diet(Harlan Laboratories™, Teklad™ diet #2918) for 2 weeks to allow foracclimatization following their arrival at UBC. In the first cohort,mice were placed on one of four different diet regimens (ResearchDiets™) for the 36-week study (n=11 per diet): (1) “10% fat” controldiet (D12450K, 10 kcal % fat, 70 kcal % carbohydrate [no sucrose]), (2)“45% fat” diet (D12451, 45 kcal % fat [primarily lard], 35 kcal %carbohydrate), (3) “60% fat” diet (D12492, 60kcal % fat [primarilylard], 20 kcal % carbohydrate), or (4) “Western” diet (D12079B, 41 kcal% fat [primarily milk fat], 43 kcal % carbohydrate [primarily sucrose]).

In the second cohort, mice were placed on either the 10% fat controldiet (D12450K; n=8) for the duration of the study of 60% fat diet(D12492; n=64) for 6 weeks, followed by one of the following treatmentregimens for the remainder of the study (n=16 per group): (1) 60% fatdiet with no drug (D12492), (2) 60% fat diet containing rosiglitazone(18 mg/kg diet or ˜3 mg/kg BW per day; Cayman Chemical™; Research Diets™custom diet formulation D08121002), (3) 60% fat diet containingsitagliptin (4 g/kg diet or ˜750 mg/kg BW per day; sitagliptin phosphatemonohydrate, BioVision™; Research Diets™ custom diet formulationD08062502R), or (4) 60% fat diet (D12492) and metformin(1,1-dimethylbiguanide hydrochloride) in drinking water (1.25 mg/ml or˜250 mg/kg BW per day).

To induce Type 1 diabetes (T1D), a single high dose injection ofStreptozotocin (STZ), for example, 190 mg/kg, (see Rezania et al. 2012),but may be anywhere above 50 mg/kg, but is typically greater than 150mg/kg. Although, a single high dose mimics the rapid and near-completedestruction of beta cells, but does lack the autoimmune component ofT1D. Alternatively, T1D may be induced a rodent with several low doseinjections of STZ to elicit an immune and inflammatory reaction, whichoften leads to the destruction of beta-cells. In contrast, a single lowdose of STZ (anywhere between 15-50 mg/kg) combined with high fat diet(HFD) is thought to better mimic the slow progression of beta-celldestruction in T2D associated with inflammation. Alternatively, a HFD(without low dose STZ) may be used to generate a model of T2D (see Bruinet al. 2015). Nevertheless, where the mice do not respond to HFD withincreased blood glucose and weight gain, a low dose of STZ may beadministered.

Numerous methods for the production of pancreatic progenitor cells fortransplantation, particularly late-stage cells, are well known in theart (see for example, Rezania, A. et al. 2014; Pagliuca, F W. et al.2014; Agulnick, A D. et al. 2015; and Russ, H A, et al. 2015).

Transplantation of hESC-Derived Pancreatic Progenitor Cells

The procedure used for transplantation of macro-encapsulated pancreaticprogenitor cells was as follows. Similar transplantation ofmacro-encapsulated pancreatic endocrine precursor cells orinsulin-producing cells was described in Rezania et al. 2014, Pagliuccaet al. 2014 and Agulnick A. D. et al. 2015. All mice were anaesthetizedwith inhalable isoflurane and transplant recipients received ˜5×10⁶hESC-derived pancreatic progenitor cells subcutaneously (s.c.) within a20 μl Theracyte™ macroencapsulation device (TheraCyte Inc.™, LagunaHills, Calif.) on the right flank, as previously described (Bruin etal., 2013). Sham mice received the same surgical procedure, but nomacroencapsulation device was implanted. In the first cohort, mice wererandomly assigned to receive either a cell transplant (Tx, n=7 per diet)or sham surgery (sham, n=4 per diet) after 7 weeks of LFD or HFDfeeding. In the second cohort, HFD-fed mice (+/− drug treatment)received either a transplant (n=8 per group) or sham surgery (n=8 pergroup) 1 week after administration of the antidiabetic drugs. LFDcontrols all received sham surgery. The treatment groups are summarizedin TABLE 2.

TABLE 2 Summary of treatment groups for in vivo transplant (Tx) studiesCohort # Diet Drug Tx/Sham Sample Size 1 10% fat None Tx 7 1 10% fatNone Sham 4 1 45% fat None Tx 7 1 45% fat None Sham 4 1 60% fat None Tx7 1 60% fat None Sham 4 1 Western None Tx 7 1 Western None Sham 4 2 10%fat None Sham 8 2 60% fat None Tx 8 2 60% fat None Sham 8 2 60% fatRosiglitazone Tx 8 2 60% fat Rosiglitazone Sham 8 2 60% fat SitagliptinTx 8 2 60% fat Sitagliptin Sham 8 2 60% fat Metformin Tx 8 2 60% fatMetformin Sham 8

Metabolic Assessments

All metabolic analyses were performed in conscious, restrained mice andblood samples were collected via the saphenous vein. BW and bloodglucose levels were assessed regularly throughout each study following a4-hr morning fast. For all other metabolic tests, blood was collectedafter fasting (time 0) and at the indicated time points followingadministration of various secretagogues.

Glucose tolerance tests (GTTs) were performed following a 6-hour morningfast and administration of glucose by oral gavage or intraperitoneal(i.p.) injection (2 glucose/kg BW, 30% solution; Vétoquinor, Lavaltrie,QC). Glucose-stimulated human C-peptide secretion from engrafted cellswas assessed following an overnight fast and an i.p. injection ofglucose (2 g/kg). Insulin tolerance tests (ITTs) were performedfollowing a 4-hour morning fast and administration of human syntheticinsulin (0.7 IU/kg body weight; Novolin ge™ Toronto, Novo Nordisk™,Mississaugua, Canada). For monthly mixed-meal challenges, mice receivedan oral gavage of Ensure™ (8 μL/g body weight; Abbott Laboratories™,Abbott Park, Ill., USA) following an overnight fast (−16 hours). Forarginine tolerance tests (ArgTT), mice received an i.p. injection ofarginine (2 g/kg, 40% solution; Sigma-Aldrich™) following a 4-hourmorning fast. Blood glucose levels were measured using a handheldglucometer (Lifescan™; Burnaby, Canada). Mouse hormone and lipidprofiles were assessed in plasma using the following kits: leptin (MouseLeptin ELISA, Crystal ChemInc.™, Downers Grove, Ill.), insulin(Ultrasensitive Mouse Insulin ELISA, Alpco Diagnostics™, Salem, N.H.),C-peptide (Mouse C-peptide ELISA, Alpco Diagnostics™), triglycerides(Serum Triglyceride kit, Sigma-Aldrich™), free fatty acids (NEFA-HR(2)kit, Wako Chemical™, Richmond, Va.) and cholesterol (Cholesterol E™ kit,Wako Chemical™). Hormone secretion from engrafted hESC-derived cells wasassessed by measuring plasma human C-peptide (C-peptide ELISA,80-CPTHU-E01.1; Alpco Diagnostics™) and human insulin and glucagonlevels (K15160C-2; Meso Scale Discovery™, Gaithersburg, Md.). HemoglobinA1c (HbA1c) levels were measured with a Siemens DCA 200 VantageAnalyzer™ (Siemens Healthcare Diagnostics™, Tarrytown, N.Y.) from wholeblood collected from the saphenous vein with EDTA as an anticoagulant.

Dual-Energy X-Ray Absorptiometry

Body composition was determined using dual-energy X-ray absorptiometry(DEXA) with a PIXImus Mouse Densitometer™ (Inside Outside Sales™). Dataare expressed as % fat.

qRT-PCR

Theracyte™ devices were harvested at 29 weeks post-transplantation fromcohort 1 and stored for qPCR analysis. The qPCR analysis, human isletdonors, and the procedure used to isolate RNA from engrafted tissue aredescribed below.

Theracyte™ devices were cut in half at the time of tissue harvest andstored in RNA Later Stabilization Solution™ (Life Technologies™,Carlsbad, Calif.) at −80° C. until use. Excess mouse tissue was firstremoved from the outside of the device before placing the device in 2 mLPBS. The edge of the device was cut off, the outer membranes peeledback, and the device isolated and placed into 400 μl Qiagen Buffer RLTPlus™ (Qiagen Inc.™, Valencia, Calif.) containing 0.1% (v/v)beta-mercaptoethanol. The PBS was collected and centrifuged at 2000×gfor 4 min to collect any cells that spilled out of the device. The cellpellet was resuspended in the same RLT Plus buffer used for lysing thecorresponding device. RNA was isolated using Qiagen RNeasy Plus Mini™kit (Qiagen Inc.™) and eluted in 16 μl nuclease-free water. RNAconcentration was measured using the NanoDrop8000 (Thermo Scientific™).Human islets were obtained from four organ donors (23-48 years of age;two males and two females) as a positive control for qPCR analysis(ProdoLabs™, Irvine, Calif.). Islet purity ranged from 85-95% andviability from 90-95%. All human islet preparations showed a 2 to 4-foldincrease in human insulin secretion after incubation with high glucoseconcentration (data not shown) using a static glucose-stimulated insulinsecretion assay, as previously described (Rezania et al., 2014).

Due to a low amount of human cells/tissue in the device, and the highprobability that some of the RNA would be from the surrounding mousetissue, the amount of human RNA was measured using a standard curve.First, all RNA was converted into cDNA using the High Capacity cDNAReverse Transcription™ kit (Thermo Fisher Scientific™/LifeTechnologies™) with the following program: 25° C. for 10 minutes, 37° C.for 2 hours, 4° C. hold. Pre-amplification was performed using a primerpool specific for the genes run (TABLE 3) and TaqMan PreAmp™ 2× MasterMix (Thermo Fisher Scientific™/Life Technologies™) with the followingcycling conditions: 95° C. 10 min, 8 cycles of 95° C. 15 s and 60° C. 4min, 99° C. 10 min, and 4° C. hold. To determine the amount of humancDNA, real-time PCR was performed on the Pre-amplified cDNA usingprimers specific to human GAPDH and mouse Gapdh and run against astandard curve made from known amounts of cDNA from a human cell line.Sixteen ng of calculated human cDNA was run on a custom TaqMan LowDensity Array™ (Thermo Fisher Scientific™/Life Technologies™; TABLE 3)using the Quant Studio 12K Flex Real Time PCR™ instrument (Thermo FisherScientific™/Life Technologies™). Data were analyzed using ExpressionSuite™ software (v1.0.3, Thermo Fisher Scientific™/Life Technologies™)and normalized to undifferentiated H1 cells using the delta delta Ctmethod. Immunofluorescent staining and image quantification to measureendogenous pancreatic beta and alpha cell area three pancreas sectionsper animal, separated by at least 200 μm, were immunostained for insulinand glucagon. Whole slide fluorescence scanning was performed using theImage Xpress Micro TM Imaging System™, and images were stitched togetherand analyzed using MetaXpress Software™ (Molecular Devices Corporation™,Sunnyvale, Calif.). The beta cell or alpha cell fraction was calculatedas the insulin-positive or glucagon-positive area/total pancreas areaand the average of three sections per animal was then multiplied by thepancreas weight. To quantify the endocrine composition within devices,the number of DAPI-positive nuclei were counted using the MultiWavelength Cell Scoring™n or both hormones was counted manually by aninvestigator who was blinded to the treatment groups. Primers are listedin TABLE 3.

TABLE 3 List of qPCR primers Gene Name Assay ID ABCC8 Hs00165861_m1 CHGBHs01084631_m1 G6PC2 Hs01549773_m1 GAPDH Hs99999905_m1 GCG Hs00174967_m1GCGR Hs01026191_g1 IAPP Hs00169095_m1 INS Hs00355773_m1 ISL1Hs00158126_m1 MAFA Hs01651425_s1 NKX6.1 Hs00232355_m1 PAX6 Hs00240871_m1PCSK1 Hs00175619_m1 PCSK2 Hs01037347_m1 SLC30A8 Hs00545183_m1 SSTHs00356144_m1 UCN3 Hs00846499_s1

Immunofluorescent Staining and Image Quantification

Prior to transplantation, a portion of differentiated pancreaticprogenitor cells were fixed overnight in 4% paraformaldehyde (PFA) andthen embedded in 1% agarose prior to paraffin embedding. In cohort 1,the Theractye™ devices and a variety of tissues (Adipose Tissue,Perirenal; Ileum; Skeletal Muscle; Cecum; Jejunum; Spleen; Colon;Kidney; Stomach, Glandular; Duodenum; Liver; Stomach, Nonglandular;Heart; Lung; and Testis) were harvested at 29 weekspost-transplantation, fixed in 4% PFA, and stored in 70% EtOH prior toparaffin embedding. All paraffin sections (5 mm thick) were prepared byWax-it Histology Services™. Immunofluorescent staining was performed aspreviously described (Rezania et al., 2011) and details about theprimary antibodies are provided in TABLE 4. H&E staining was performedaccording to standard procedures and tissue analysis was performed in ablinded fashion by an independent pathologist (Nova Pathology PC™).

TABLE 4 Antibody information for immunofluorescent staining AntigenSpecies Source Dilution CK19 Mouse Dako ™; Denmark 1:100 C-PeptideGuinea Pig Abcam ™; Cambridge, MA 1:100 F4/80 Rat AbD Serotec ™;Kidlington, UK 1:100 FGF21 Rabbit Abcam ™; Cambridge, MA 1:50  Glucagon(Ms) Mouse Sigma-Aldrich ™; St Louis, MO  1:1000 Glucagon (Rb) RabbitCell Signaling ™; Danvers, MA 1:500 Insulin (GP) Guinea PigSigma-Aldrich ™; St Louis, MO  1:1000 Insulin (Rb) Rabbit CellSignaling ™; Danvers, MA 1:100 Insulin (MAb1) Mouse Millipore ™;Billerica, MA 1:200 MAFA Rabbit Custom Antibody; Lifespan Biosciences ™; 1:1000 Seattle, WA NKX6.1 Rabbit Custom Antibody; LifespanBiosciences ™;  1:1000 Seattle, WA NKX2.2 Mouse Developmental StudiesHybridoma Bank ™; 1:100 University of Iowa; Iowa City, IA PAX6 RabbitCovance ™; Princeton, NJ 1:250 PCNA Mouse BD Biosciences ™; Mississauga,ON 1:100 PDX1 Guinea Pig Abcam ™; Cambridge, MA  1:1000 Somatostatin(Ms) Mouse Sigma-Aldrich ™; St Louis, MO 1:100 Somatostatin (Rb) RabbitSigma-Aldrich ™; St Louis, MO 1:500 Synaptophysin Rabbit NovusBiologicals ™; Littleton, CO 1:50  Trypsin Sheep R&D Systems ™;Minneapolis, MN 1:100

Statistical Analysis

All statistical analyses were performed using GraphPad Prism™ software(GraphPad Software™). Two-way repeated measure ANOVAs were performedwith a Fisher's LSD post-hoc test to compare HFD mice with LFD controlsat different time points. One-way repeated measures ANOVA were performedwith Dunnett post-hoc to compare values at different time points tobaseline levels (time 0) within each treatment group. One-way ANOVAswere performed with a Dunnett post-hoc test for multiple comparisons to10% fat controls or a Student-Neuman-Keuls test to compare betweenmultiple groups. The qPCR data was assessed by one-way ANOVA with aFisher's LSD post-hoc test to compare grafts from various treatmentgroups with either human islets or HFD-fed mice without drug treatment.Unpaired t-tests were used to compare the effect of transplantationwithin a single treatment group (i.e. sham vs tx) and paired t-testswere used when comparing samples pre-and post-administration of ainsulin or glucagon secretogogue (glucose, oral meal, arginine). Areaunder the curve was calculated with y=0 as the baseline. For ITTs, areaabove the curve was calculated using the fasting blood glucose level foreach animal as the baseline. For all analyses, p<0.05 was consideredstatistically significant. Data are presented as the mean±SEM (linegraphs) or as box-and-whisker plots showing individual data points.

Cell Preparation for SH-1533: Advance Differentiation of S4D4 IDP toAdvance Day 9

S4D4 IDP cells were thawed into each of two 500 ml PBS-MINI verticalspinners with 400 ml Stage 5 thaw media per spinner. To ensure adequatedilution of DMSO in the cell suspension, while cells were settled on thebottom of the vial, approximately 3 ml of cryopreservation media wasaspirated from each vial prior to transfer of the cells to the PBS-MINIspinner. Subsequently, thaw media was added drop wise to the spinner todilute the population. The thaw media consisted of DMEM-HG mediasupplemented with 2% KSR, 1:200 ITS-X, 10 ug/ml Heparin, 100 nM LDN, 1uM T3, 5 uM ALK5i, 250 nM SANT-1, 50 nM RA, 10 uM Y-27632, and 4 ku/mlDNase. Each vessel was placed on a PBS-MINI base in the BSC set to 25RPM and 2×5 ml samples were pulled for cell counts using the accutasecount method and the NC100 NucleoCounter. The vessels were then culturedovernight in a 37° C. 5% CO2 incubator on a PBS-MINI base set to 20 RPM.

Following overnight culture each vessel containing cells was placed inthe BSC for ˜5-10 minutes to allow cells to settle to the bottom of thespinner. The majority of the spent culture media was aspirated from thevessel and fresh Stage 5 media containing DMEM-HG media supplementedwith 2% KSR, 1:200 ITS-X, 10 ug/ml Heparin, 100 nM LDN, 1 uM T3, 5 uMALK5i, 250 nM SANT-1, and 50 nM RA. Each vessel was placed on a PBS-MINIbase in the BSC set to 25 RPM and 2×5 ml samples were pulled for cellcounts using the accutase count method and the NC100 NucleoCounter. Thecultures were then again cultured overnight as described above exceptthat the speed of the base was increased to 22 RPM to help decreaseclumping of clusters.

On the third day of culture, the media was changed to Stage 6 mediacontaining all Stage 5 components plus the addition of 100 nM gammasecretase inhibitor (XX). The culture continued with daily stage 6 mediaexchanges until S6D7 complete at which time the cells were transferredto a perfusion spinner for washing and aliquoting. Cells were aliquotedinto 5million cells/aliquot using the sampling port on the spinner flaskand a wee syringe and then transferred to a 1.5 ml centrifuge tube forkidney capsule transplant. Kidney capsule aliquots were transferredimmediately to the transplant site via courier at ambient temperature ina DMEM-HG basal media supplemented with 2% KSR, 1:200 ITS-X, and 10ug/ml Heparin. A biomarker expression check was done on the cellpopulation at S6D7 via FACS, PCR, IHC, and phase contrast imaging.Sterility samples were collected from the reagents and the residualmedia from all kidney capsule aliquots prior to transplantation.

FIGS. 12, 13—Male and female SCID-Beige mice(C.B-Igh-1b/GbmsTac-Prkdc^(scid)-Lyst^(bg)N7; Taconic, Hudson, N.Y.)received ad libitum access to a standard irradiated diet (Teklad Diet#2918—Harlan Laboratories, Madison, Wis., USA) and were maintained on a12 h light/dark cycle throughout the study. At 7 weeks old, all micewere anaesthetized with inhalable isoflurane and transplant recipientsreceived either ˜5×10⁶ stage 4 cells (5M S4), ˜1×10⁶ stage 4 cells (1MS4), or ˜1×10⁶ stage 7 cells (1M S7) under the kidney capsule on theleft flank: 5M S4 females (N=10), 5M S4 males (N=11), 1M S4 males (N=5),1M S7 females (N=9), 1M S7 males (N=9). Differentiated human ES cellswere produced using the methods described in Rezania, A. et al. 2014.Body weight was assessed bi-weekly or monthly throughout the studyfollowing a 4-hour morning fast.

FIG. 14—Six week old immunocompromised SCID Beige mice were acclimatizedfor 1 week post arrival. Animals were then injected with a single doseof 190 mg/kg of the beta-cell toxin streptozotocin (STZ) to induce amodel of type 1 diabetes. 1.5 Million Stage 7 cells produced using themethods described in Rezania, A. et al. 2014, or ˜6000 isolated humanislet equivalents (IEQs) were transplanted under the kidney capsule.Control mice received neither STZ nor cell transplants. Mice weresubsequently monitored weekly for 4h fasting blood glucose levels andbody weight. Human islet recipient mice consisted of mice that hadpreviously been injected with STZ but returned to normoglycemia, exceptfor one animal that was hyperglycemic, in addition to non-STZ treatedmice.

FIG. 15A—Blood for glucose measurements and sampling were from thesaphenous vein of conscious restrained mice. For hormone detection bloodwas collected in heparinized capillaries, transferred on ice into 1.5 mltubes and the plasma separated from blood cells after a 9 min spin at7000 rpm. Samples were stored at −30° C. before assaying for leptin byELISA.

FIGS. 15B to 17—Body composition of mice was measured on the dayfollowing metabolic cage analysis via dual energy X-ray absorptiometry(DEXA) measurements on isoflurane anesthetized mice. Fat pad and organweights were determined by dissection and immediate weighing of tissues.

FIG. 18—To differentiate cryostored Stage 4 cells to Stage 6 cells,cells were thawed into each of two 500 ml PBS-MINI vertical spinnerswith 400 ml Stage 5 thaw media per spinner. To ensure adequate dilutionof DMSO in the cell suspension, while cells were settled on the bottomof the vial, approximately 3 ml of cryopreservation media was aspiratedfrom each vial prior to transfer of the cells to the PBS-MINI spinner.Subsequently, thaw media was added drop wise to the spinner to dilutethe population. The thaw media consisted of DMEM-HG media supplementedwith 2% KSR, 1:200 ITS-X, 10 μg/ml Heparin, 100 nM LDN, 1 μM T3, 5 μMALK5i, 250 nM SANT-1, 50 nM RA, 10 μM Y-27632, and 4 ku/ml DNase. Eachvessel was placed on a PBS-MINI base in the BSC set to 25 RPM and 2×5 mlsamples were pulled for cell counts using the accutase count method andthe NC100 NucleoCounter. The vessels were then cultured overnight in a37° C. 5% CO2 incubator on a PBS-MINI base set to 20 RPM. Followingovernight culture each vessel containing cells was placed in the BSC for˜5-10 minutes to allow cells to settle to the bottom of the spinner. Themajority of the spent culture media was aspirated from the vessel andfresh Stage 5 media containing DMEM-HG media supplemented with 2% KSR,1:200 ITS-X, 10 μg/ml Heparin, 100 nM LDN, 1 μM T3, 5 μM ALK5i, 250 nMSANT-1, and 50 nM RA. Each vessel was placed on a PBS-MINI base in theBSC set to 25 RPM and 2×5 ml samples were pulled for cell counts usingthe accutase count method and the NC100 NucleoCounter. The cultures werethen again cultured overnight as described above except that the speedof the base was increased to 22 RPM to help decrease clumping ofclusters. On the third day of culture, the media was changed to Stage 6media containing all Stage 5 components plus the addition of 100 nMgamma secretase inhibitor (XX). The culture continued with daily stage 6media exchanges until S6D7 complete at which time the cells weretransferred to a perfusion spinner for washing and aliquoting. Cellswere aliquoted for transplant.

Cell transplants were either under the kidney capsule or in asubcutaneous deviceless pocket as described by Pepper, A R. et al. 2015Specifically 4 weeks before cell transplant, 2-cm segments of a 5-French(Fr.) textured nylon radiopaque angiographic catheter were implantedsubcutaneously into the lower left quadrant of SCID Beige mice. A 4-mmlateral transverse incision was made caudal to the rib cage allowing fora small pocket to be created inferior to the incision line using bluntdissection. An adequate void (1 cm by 3 cm) was created. The cathetersegment was implanted into the space such that the catheter laidparallel to the midline. The incision was sutured closed. Onceimplanted, the catheter became adherent with blood proteins, leading tothe formation of densely vascularized tissue, which exhibited aminimally visible profile. At the time of transplant, removal of thecatheter revealed a vascularized lumen allowing for cellular transplantinfusion.

Deviceless-recipient mice were maintained under anesthesia with inhalantisoflurane and placed in a supine position. A field surrounding theimplanted catheter was prepared by shaving and disinfecting the surface.Cranial to the superior edge of the implanted catheter, a small (4 mm)incision was made to gain access to the catheter. The tissue matrixsurrounding the superior margin of the catheter was dissected towithdraw and remove the catheter. The cells were then delivered into thespace using a pipette tip. The incision was sutured closed. Prior torecovery, recipients received a 0.1 mg/kg subcutaneous bolus ofbuprenorphine.

Control animals received the same dose of cells under the kidneycapsule, the standard site for rodent islet transplantation. For allexperiments cells were pooled, batched and transplanted in randomallocation to either the DL or KC sites. To facilitate the KCtransplants, a left lateral para-lumbar subcostal incision was made andthe left kidney was delivered into the wound. The renal capsule wasincised and space was made under the capsule to allow transplantation ofthe cells using PE-50 tubing. The subcostal incision was closed in twolayers.

Animals were monitored at regular intervals for body weight, and 4 hourfasting glycemia. Blood was collected from the saphenous vein of randomfed mice 2, 6 and 10 weeks post transplant and plasma was assayed forhuman C-peptide by ELISA (Alpco).

The invention will be further clarified by a consideration of thefollowing, non-limiting examples.

EXAMPLES

All metabolic analyses used in these Examples were performed using bloodsamples collected via saphenous vein. Body weight and blood glucoselevels were assessed regularly throughout each study following a 4-hourmorning fast. For all other metabolic tests, blood was collected afterfasting (time zero) and at the indicated time points followingadministration of various secretagogues. Body composition was determinedusing dual-energy X-ray absorptiometry (“DEXA”) with a PIXImus MouseDensitometer™ (Inside Outside Sales™, Madison, Wis.). Data are expressedas % fat.

Example 1: Development of a Model of Obesity and Type 2 Diabetes (T2D)in Immunodeficient Mice

For purposes of carrying out the examples, 8 to 10 week old male,SCID-beige mice (C.B-Igh-1b/GbmsTac-Prkdc^(scid)-Lyst^(bg)N7; Taconic™,Hudson, N.Y.) were maintained on a 12 hour light/dark cycle. All micereceived ad libitum access to a standard irradiated diet (HarlanLaboratories™, Teklad Diet™ #2918, Madison, Wis.) for 2 weeks to allowfor acclimatization. Mice were placed on one of four different dietregimens (Research Diets™, New Brunswick, N.J., USA) for the 36 weekstudy (n=11 per diet): 1) “10% fat” control diet (D12450K—10 kcal % fat;70 kcal % carbohydrate, no sucrose); 2) “45% fat” diet (D12451—45 kcal %fat, primarily lard; 35 kcal % carbohydrate); 3) “60% fat” diet(D12492—60 kcal % fat, primarily lard; 20 kcal % carbohydrate); or 4)“western” diet (D12079B—41 kcal % fat, primarily milk fat; 43 kcal %carbohydrate, primarily sucrose).

Blood glucose levels were measured using a handheld glucometer(Lifescan™, Milpitas, Calif.). Mouse hormone and lipid profiles wereassessed in plasma using the following kits: leptin (Mouse Leptin ELISA,Crystal Chem Inc.™, Downers Grove, Ill.), insulin (Ultrasensitive MouseInsulin ELISA, Alpco Diagnostics™, Salem, N.H.), C-peptide (MouseC-peptide ELISA, Alpco Diagnostics™), triglycerides (Serum Triglyceride™kit, Sigma-Aldrich™), free fatty acids (NEFA-HR(2) kit, Wako Chemical™,Richmond, Va.) and cholesterol (Cholesterol E kit™, Wako Chemical™).Hormone secretion from engrafted hESC-derived cells was assessed bymeasuring plasma human C-peptide (C-peptide ELISA, 80-CPTHU-E01.1; AlpcoDiagnostics™) and human insulin and glucagon levels (K15160C-2; MesoScale Discovery™, Gaithersburg, Md.). Hemoglobin A1c (HbA1c) levels weremeasured with a Siemens DCA 200 Vantage Analyzer™ (Siemens HealthcareDiagnostics™, Tarrytown, N.Y.) from whole blood collected from thesaphenous vein with EDTA as an anticoagulant.

FIG. 1 and FIG. 7 display body weight gain, measures of glucosehomeostasis and adipocyte characterization in mice on the various diets.All three high fat diets (HFD; 45% fat, 60% fat, and western) inducedrapid increases in fasting body weight (FIG. 1A) and blood glucoselevels (FIG. 1B) compared to low fat diet (LFD) controls (10% fat).Moreover, after five days, mice in all three HFD groups were severelyglucose intolerant relative to LFD controls (FIG. 7A), prior todifferences in body weight (FIG. 7B). At 32 days, HFD mice were bothglucose intolerant (FIG. 7C) and significantly heavier (FIG. 7D) thanLFD controls. Mice fed 45% and 60% fat diets were overtly insulinresistant at day 42 (higher glucose levels at 10 and 60-120 minutespost-insulin, and reduced area above the curve relative to LFDcontrols), whereas Western diet mice only showed significant insulinresistance at 10 minutes after insulin administration (FIG. 1E).

Mice in all HFD groups developed glucose intolerance (day 47, FIG. 1C)and had insulin secretion kinetics that differed from LFD controls(either no glucose-induced insulin secretion or altered timing of peakinsulin levels; FIG. 1D). All HFD mice were significantly overweight(FIG. 1A) and had increased adiposity (FIG. 1F) compared to LFDcontrols; mice fed 45% and 60% fat diets also had significantly elevatedcirculating leptin levels (FIG. 1G). Exposure to HFDs causeddyslipidemia, including significantly reduced plasma free fatty acidlevels in all HFD-fed mice (FIG. 7E), reduced triglyceride levels in 45%and 60% fat groups (FIG. 7F), and elevated cholesterol levels in thewestern diet group compared to LFD controls (FIG. 7G). The HFD-inducedmetabolic defects in immune-deficient mice were not associated withmacrophage infiltration in adipose tissue (marked by F4/80immuno-reactivity, whereas significant accumulation of F4/80-positivecrown-like structures were observed in the epididymal fat of ob/ob mice,an immunocompetent model of T2D (micrographs not shown). Theimmuno-fluorescent staining of epididymal fat from the mice of Example 1fed either 10% or 60 % fat diets for 36 weeks and of an ob/ob mouse wascarried out, wherein F4/80 and FGF21 are, respectively, a macrophage andan adipocyte markers.

Example 2: In Vitro Generation of Pancreatic Endocrine Progenitor Cellsfrom Human Embryonic Stem Cells

Cells of the human embryonic stem cell line H1 (WA01 cells, WiCellResearch Institute™, Madison, Wis.) were seeded as single cells at 1×10⁵cells/cm² on 1:30 diluted MATRIGEL™ (Becton Dickinson BioSciences™,Franklin Lakes, N.J.; Catalogue (“Cat.”) No. 356231) coated dishes inmTeSR-1™ (Stem Cell Technologies™, Vancouver, BC; Cat. no. 05850). At˜70-80% confluency, the H1 cell cultures were rinsed with 1× Dulbeccos'sphosphate buffered saline without Mg2+ and Ca2+ (Invitrogen™, Carlsbad,Calif.; Cat. No.14190) followed by incubation with 0.02% Versene™(“EDTA”) (Lonza™, Walkersville, Md.; Cat. No. 17-711E) for 12 mins atroom temperature. Released single cells were rinsed with mTeSR-1™, andspun at 1000 rpm for 5 mins. The resulting cell pellet was re-suspendedin mTeSR-1™ medium supplemented with 10 μM of the ROCK inhibitorY-27632™ (Sigma-Aldrich™, St. Louis Miss.; Cat. No. Y0503) and thesingle cell suspension was seeded at approximately 1.3×10⁵ cells/cm².Cultures were fed every day and differentiation was initiated 48 hrsfollowing seeding, resulting in ˜90% starting confluency. The cultureswere differentiated using the following protocol.

-   -   Stage 1 (3 days): Undifferentiated H1 cells plated on MATRIGEL™        coated surfaces (90% confluent) were exposed to RPMI 1640™        medium (Invitrogen™, Cat. No. 22400) supplemented with 1.2 g/L        sodium bicarbonate (Sigma-Aldrich™, Cat. No. S6297), 0.2% fetal        bovine serum (“FBS”) (Hyclone™, South Logan, Utah; Cat. No.        SH30071.02), 100 ng/mL activin-A (“AA”) (Peprotech™, Rocky Hill,        N.J.; Cat. No. 338-AC-010), and 20 ng/mL of Wnt3A (R&D Systems,        Inc.™, Minneapolis, Minn.; Cat. No. 5036-WN) for day one only.        For the next two days, cells were cultured in RPMI with 0.5%        FBS, 1.2 g/L sodium bicarbonate, and 100 ng/mL AA.    -   Stage 2 (3 days): Stage 1 cells were cultured in DMEM-F12 medium        (Invitrogen™ (Gibco™); Cat. No. 10565-018) supplemented with 2        g/L sodium bicarbonate, 2% FBS and 50 ng/mL of FGF7 (Peprotech™,        Cat. No. 100-19) for three days.    -   Stage 3 (4 days): Stage 2 cells were cultured in DMEM-HG (high        glucose) medium (Invitrogen™. Cat. No. 10569-044) supplemented        with 0.25 μM SANT-1        (N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazineamine)        (Sigma-Aldrich™, Cat. No. S4572), 2 μM retinoic acid (“RA”)        (Sigma-Aldrich™, Catalog No. R2625), 100 ng/mL of Noggin™ (R&D        Systems™, Cat. No. 6057-NG), and 1% (v/v) B27 (Invitrogen™        (Gibco™), Cat. No. 17504-044).    -   Stage 4 (5 days): Stage 3 cells were cultured for 4 days in        DMEM-HG medium supplemented with 0.1 μm        2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine        (“ALK5 inhibitor II”, “ALK5i”) (Axxora™, San Diego, Calif.; Cat.        No. ALK-70-445), 100 ng/mL Noggin, 500 nM        (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam        (“TPB”) (Shanghai ChemPartner Co., LTD™, China) and 1% B27. For        the last day of culture, cells were treated with 5 mg/mL        Dispase™ for 5 min at 37° C., followed by gentle pipetting to        break the cells into cell clusters (<100 μm). The cell clusters        were transferred into a polystyrene 125-500 ml Spinner Flask        (Corning™), and spun at 80-100 rpm overnight in suspension with        DMEM-HG supplemented with 0.2 μM ALK5i, 100 nM        (6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine,        hydrochloride)) (“LDN”) a BMP receptor inhibitor (Stemgent™, San        Diego, Calif.; Cat. No. 04-0074) and 1% B27.

As shown in (FIG. S2), the resulting pancreatic endocrine precursorcells were assessed by fluorescence-activated flow cytometry (“FACS”)and immuno-fluorescent staining (micrographs not shown). FACS stainingwas conducted as described in Diabetes, 61, 2016, 2012 and using theantibodies listed in TABLE 5. In brief, cells were incubated in TrypLE™Express (Life Technologies™, Catalog No. 12604) for 3-5 minutes at 37°C. and released into single cell suspensions after which they werewashed twice with a staining buffer of PBS containing 0.2% BSA (BDSciences™, Cat. No. 554657). Cells (1×10⁵ to 1×10⁶) were resuspended in100 μl blocking buffer of 0.5% human gamma globulin diluted 1:4 instaining buffer for surface marking. Added to the cells at a finaldilution of 1:20 were directly conjugated primary antibodies followed byincubation at 4° C. for 30 minutes. The stained cells were twice washedin the staining buffer, followed by re-suspension in 200 μl stainingbuffer and then incubated in 15 μl of 7-AAD for live/dead discriminationbefore FACS analysis on the BD Canto II. Intracellular antibody stainingwas accomplished by first incubating with Green Fluorescent LIVE/DEADcell dye (Life Technologies™, Cat. No. L23101) at 4° C. for 20 minutesfollowed by a single wash in cold PBS. Fixing of cells was in 250 μl ofCytoflx/Cytoperm™ buffer (BD Sciences™, Cat. No. 554723) followed byre-suspension of the cells in 100 μl of Perm™ wash bufferstaining/blocking solution with 2% normal goat serum. Cells wereincubated at 4° C. for 30 minutes with primary antibodies at empiricallypre-determined dilutions followed by two washes in Perm/Wash buffer.Cells were then incubated with the appropriate antibodies at 4° C. for30 minutes and then washed twice prior to analysis on the BD FACS CantoII™. The concentrations of antibodies used are shown on TABLE 5. Theantibodies for pancreas markers were tested for specificity using humanislets or undifferentiated H1 cells as a positive control. For secondaryantibodies, the following were added and incubated at 4° C. for 30minutes: anti-mouse Alexa Fluor™ 647 at 1:500 (Life Technologies™), goatanti-rabbit PE at 1:200 (v) or donkey anti-goat Alexa 647™ at 1:800(Life Technologies™) followed by a final wash in perm Wash buffer andanalysis on BD FACS Canto II using BD FACS Diva Software™ with at least30,000 events being acquired.

Following in vitro differentiation, 98.8% of cells expressed PDX1 and71.7% expressed NKX6.1 (FIGS. 8A, 8C, 8D and 8G), two key markers ofpancreatic endoderm. Approximately 20% of PDX1-positive cells were inthe cell cycle, as indicated by Ki67 or PCNA expression (FIGS. 8A and8D), and the pluripotency marker OCT3/4 was not detected (FIG. 8A).Although ˜16% of progenitor cells expressed endocrine markers (FIGS. 8Aand 8B), only 2.8% of synaptophysin-positive cells co-expressed NKX6.1(FIG. 8A) and most were polyhormonal (FIG. 8F), indicative of animmature endocrine population. Insulin/C-peptide-positive cells onlyrarely co-expressed PAX6 (FIG. 8E) or NKX6.1 (FIG. 8C) at this stage ofdifferentiation.

TABLE 5 List of Antibodies used for FACS analysis Antigen SpeciesSource/Catalogue Number Dilution Glucagon Mouse Sigma-Aldrich ™/G2654 1:250 Insulin Rabbit Cell Signaling Technology Inc. ™, Danvers. 1:10MA/3014B NKX6.1 Mouse Developmental Studies Hybridoma Bank ™ 1:50 IowaCity, Iowa/F55A12 NKX2.2 Mouse Developmental Studies Hybridoma  1:100Bank/74.5A5 PDX1 Mouse BD BioSciences ™, San Jose, CA/562161 1:50 Ki67Mouse BD Biosciences ™, 558595 1:20 Pax6 Mouse BD Biosciences ™, 5615521:20 Chromogranin A Rabbit Dako, Carpinteria ™, CA/A0430 1:40 ISL-1Mouse BD Biosciences ™, 562547 1:20 NeuroD Mouse BD Bioscience ™, 5630011:40 FOXA2 Mouse BD Bioscience ™, 561589 1:80 OCT3/4 Mouse BDBiosciences ™, 560329 1:20

Example 3: Exposure to HFDs Did Not Affect the Function of hESC-DerivedEndocrine Cells In Vivo

The pancreatic endocrine precursor cells of Example 2 were encapsulatedwithin a 20 μl Theracyte™ macro-encapsulation device (TheraCyte Inc.™,Laguna Hills, Calif.) as follows. Approximately 5×10⁶ endocrineprecursor cells (in cluster form) were placed into a positivedisplacement pipette. Using slight pressure, the tip of thecapillary/piston tip containing cells was placed snug in the hub of the24 gauge catheter and the cells dispensed from the positive displacementpipette through the catheter into the device. The device was sealedusing a titanium barb. The encapsulated pancreatic endocrine precursorcells were then transplanted subcutaneously into seven SCID-beige micefrom each of the four diet regimens. All mice were anaesthetized withinhalable isoflurane and transplant recipients received approximately5×10⁶ pancreatic endocrine precursor cells subcutaneously on the rightflank. Four sham mice received the same surgical procedure, but nomacro-encapsulation device was implanted.

Following transplantation, the pancreatic endocrine precursor cellsfurther differentiated in vivo and the resulting cells, from all dietgroups, secreted similar levels of human C-peptide under basal and fedconditions between 8 and 20 weeks (FIG. 2A) and produced robustglucose-stimulated human C-peptide secretion at 18 weeks (FIGS. 2B,C).Similarly, human insulin secretion was induced by an arginine challengein all diet groups at 24 weeks (FIG. 2D). A trend towards increasedbasal glucagon secretion in the HFD groups was observed, but becausefour out of five mice in the LFD group had undetectable fasting glucagonlevels, it was not possible to do a statistical analysis (FIG. 2E).Arginine-stimulated glucagon levels were similar between diet groups(FIGS. 2E,F) and it was estimated that approximately half of thecirculating glucagon may have originated from the hESC-derived cells, asindicated by the difference between transplanted and sham glucagonlevels (FIG. 2F).

The Theracyte™ devices were cut in half at the time of tissue harvestand stored in RNAlater Stabilization Solution™ (Qiagen, Inc.™, Valencia,Calif.; Cat. No.76106) at −80° C. until use. Excess mouse tissue wasfirst removed from the outside of the device before placing the devicein 2 mL PBS. The edge of the device was cut off, the outer membranespeeled back, and the device isolated and placed into 400 μl Qiagen™Buffer RLT Plus (Qiagen Inc.™, Cat. No.79216) containing 0.1% (v/v)beta-mercaptoethanol. The PBS was collected and centrifuged at 2000×gfor 4 min to collect any cells that spilled out of the device. The cellpellet was re-suspended in the same RLT Plus buffer used for lysing thecorresponding device. RNA was isolated using Qiagen RNeasy Plus Mini™kit (Qiagen Inc.™; Cat. No74316) and eluted in 16 μl nuclease-freewater. RNA concentration was measured using the NanoDrop8000™ (ThermoScientific™).

Human islets were obtained from four organ donors (23-48 years of age;two males and two females) as a positive control for quantitativepolymerase chain reaction (“qPCR”) analysis (Prodo Labs; Irvine,Calif.). Islet purity ranged from 85-95% and viability from 90-95%. Allhuman islet preparations showed a 2 to 4-fold increase in human insulinsecretion after incubation with high glucose concentration (data notshown) using a static glucose-stimulated insulin secretion assay. Inbrief, human islet cells (approximately 20 to 50 islet cells) wererinsed twice with Krebs buffer (129 mM NaCl, 4.8 mM KCL, 2.5 mM CaCl₂,1.2 mM MgSO₂. 1 mM Na₂HPO₄, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES, and0.1% BSA in deionized water and then sterile filtered) and thenpre-incubated in Krebs buffer for 40 mins. Cells were then incubated inKrebs buffer spiked with 3.3 mM glucose for 60 mins. The cells were thentransferred to another plate containing Krebs buffer spiked with 16.7 mMglucose and incubated for an additional 60 mins. Supernatant sampleswere collected after each incubation period and frozen at −70° C. forhuman C-peptide ELISA (Mercodia™, Winston-Salem, N.C.; Cat. No.10-1141-01) measurement.

Due to a low amount of human cells/tissue in the device, and the highprobability that some of the RNA would be from the surrounding mousetissue, the amount of human RNA was measured using a standard curve.First, all RNA was converted into cDNA using the High Capacity cDNAReverse Transcription Kit™ (Thermo Fisher Scientific™/LifeTechnologies™) with the following program: 25° C. for 10 minutes, 37° C.for 2 hours, 4° C. hold. Pre-amplification was performed using a primerpool specific for the genes run (TABLE 3) and TaqMan PreAmp 2× MasterMix™ (Thermo Fisher Scientific™/Life Technologies™) with the followingcycling conditions: 95° C. 10 min, 8 cycles of 95° C. 15 s and 60° C. 4min, 99° C. 10 min, and 4° C. hold.

To determine the amount of human cDNA, real-time PCR was performed onthe Pre-amplified cDNA using primers specific to human GAPDH and mouseGapdh and run against a standard curve made from known amounts of cDNAfrom a human cell line. Sixteen ng of calculated human cDNA was run on acustom TaqMan Low Density Array™ (Thermo Fisher Scientific™/LifeTechnologies™; TABLE 3) using the Quant Studio 12K Flex Real Time PCR™instrument (Thermo Fisher Scientific/Life Technologies). Data wereanalyzed using Expression Suite™ software (v1.0.3, Thermo FisherScientific™/Life Technologies™) and normalized to undifferentiated H1cells using the delta delta Ct method.

Prior to transplant, a portion of the pancreatic endocrine precursorcells were fixed overnight in 4% paraformaldehyde (“PFA”) and thenembedded in 1% agarose prior to paraffin-embedding. Theractye™ devices,as well as a variety of tissues (listed in TABLE 3 above), wereharvested at 29 weeks post-transplant, fixed in 4% PFA and stored in 70%ethyl alcohol prior to paraffin-embedding. All paraffin sections (5 μmthickness) were prepared by Wax-it Histology Services™ (Vancouver, BC).Primary antibodies are provided in TABLE 4, above. Hemotoxyline andeosin (“H&E”) staining was performed using standard procedures andtissue analysis was performed in a blinded fashion by an independentpathologist (Nova Pathology PC™, Bellingham, Wash., USA).

At 29 weeks post-transplant, the encapsulated hESC-derived grafts hadsimilar or significantly higher levels of islet-related genes comparedto human islets and there were no significant differences amongdifferent LFD or HFD groups (data not shown—CHGB; INS; CGC; SST; NKX6.1;PAX6; ISL1; MAFA; ABCC8; IAPP; PCSK1; PCSK2; GCGR; G6PC2; SLC30A8; andUCN3). In particular, genes CHGB, INS, CGC, SST, MAFA and PCSK1, hadsimilar gene expression in encapsulated hESC-derived grafts (i.e. for10% fat; 45% fat; 60% fat and Western diets) when compared to humanislets. However, when NKX6.1, PAX6, ISL1, ABCC8, IAPP, PCSK2, GCGR,G6PC2, SLC30A8 and UCN3 gene expression in encapsulated hESC-derivedgrafts (i.e. for 10% fat; 45% fat; 60% fat and Western diets) wascompared to human islets, the hESC-derived graft cells generally hadsignificantly higher levels of gene expression than the human islets.

The majority of cells within the harvested devices were immuno-reactivefor the endocrine marker synaptophysin, and a small proportion expressedthe ductal marker CK19; trypsin-positive exocrine cells were rarelyobserved (micrographs not shown). The grafts were largely composed ofcells expressing either insulin, glucagon or somatostatin, and thepercentage of mono-hormonal insulin-positive and glucagon-positive cellswas similar between diet groups (FIG. 3). A minor, but significantlyhigher percentage of cells that were immuno-reactive for both insulinand glucagon in the HFD grafts compared to LFD grafts (FIG. 3) werenoted. Aside from these rare polyhormonal cells, exposure to HFDs didnot appear to generally influence the maturation state of hESC-derivedinsulin-secreting cells; the majority of insulin-positive cells in alltransplant recipients co-expressed PDX1, NKX2.2, NKX6.1, and MAFA at 29weeks post-transplant (micrographs not shown).

Example 4: hESC-Derived Insulin Secreting Cells Improved Diet-InducedDysglycemia and Insulin Resistance

Glucose tolerance tests (“GTTs”) were performed at 18 and 24 weekspost-transplant following a 6-hour morning fast and administration ofglucose by oral gavage or intraperitoneal (“i.p.”) injection (2 gglucose/kg BW, 30% solution; Vétoquinol™, Lavaltrie, QC).Glucose-stimulated human C-peptide secretion from engrafted cells wasassessed following an overnight fast and an i.p. injection of glucose (2g/kg). Insulin tolerance tests (“ITTs”) were performed 22weeks-post-transplant following a 4-hour morning fast and administrationof human synthetic insulin (0.7 IU/kg body weight; Novolin ge™ Toronto,Novo Nordisk™, Mississauga, Canada). For monthly mixed-meal challenges,mice received an oral gavage of Ensure™ (8 uL/g body weight; AbbottLaboratories™, Abbott Park, Ill., USA) following an overnight fast (˜16hours). For arginine tolerance tests (“ArgTT”), mice received an i.p.injection of arginine (2 g/kg, 40% solution; Sigma-Aldrich™) following a4-hour morning fast.

All HFD groups continued to be overweight and hyperglycemic underfasting conditions compared to LFD controls throughout the duration ofthe study. Transplantation of the encapsulated cells did not affecteither body weight or fasting blood glucose levels compared to shamsurgery (data not shown). However, significant improvements in long-termglycemic control, as measured by HbA1C, following transplantation alone(FIGS. 4A and B) was observed. HbA1C levels were elevated at 12 and 24weeks in all HFD sham mice compared to LFD sham controls andsignificantly reduced by transplantation in the 45-60% fat group at bothages (FIGS. 4A and 4B). Transplant recipients on 45-60% fat diets alsodisplayed a significantly lower glucose excursion following a mixed-mealstimulus compared to sham mice at 20 weeks (FIG. 4C) and all HFDtransplant recipients had significantly improved glucose tolerance at 24weeks post-transplant (FIG. 4E, FIG. 9B); these improvements were notyet evident at 18 weeks (FIG. 4D, FIG. 9A). Glucose tolerance in the45-60% group was not completely ameliorated at 24 weeks, but transplantrecipients in the western group had an area under the curve that wasindistinguishable from controls (FIG. 4E; FIG. 9B). A significantimprovement in insulin sensitivity at 22 weeks in transplanted HFD-fedmice compared to shams (FIG. 4F; FIGS. 9C and 9D) was also observed,which may have contributed to the improved glucose tolerance in HFDtransplant recipients (FIG. 4E).

To measure endogenous pancreatic beta and alpha cell area three pancreassections per animal, separated by at least 200 μm, were immuno-stainedfor insulin and glucagon. Whole slide fluorescence scanning wasperformed using the ImageXpress Micro Imaging System™, and images werestitched together and analyzed using MetaXpress Software™ (MolecularDevices Corporation™, Sunnyvale, Calif.). The beta cell or alpha cellfraction was calculated as the insulin-positive or glucagon-positivearea/total pancreas area and the average of three sections per animalwas then multiplied by the pancreas weight. To quantify the endocrinecomposition within devices, the number of DAPI-positive nuclei werecounted using the Multi Wavelength Cell Scoring™ module in MetaXpress™and the number of cells that were immuno-reactive for insulin, glucagonor both hormones was counted manually by an investigator who was blindedto the treatment groups.

Beta cell mass was significantly higher in all mice on 60% fat dietscompared to LFD sham controls, and there was no difference between shamand transplanted mice in either diet group (FIG. 10A). There was noeffect of HFDs on alpha cell mass, but a significant reduction in alphacell mass was observed in LFD transplant recipients compared to LFDshams (FIG. 10B). There were no significant differences in the ratio ofinsulin-positive to glucagon-positive area in the pancreas of mice oneither diet (FIG. 10C).

Example 5: Cell Therapy Alone had No Effect on the Obesity Phenotype

Although the encapsulated hESC-derived cells improved glucosehomeostasis in HFD-fed mice, there was no apparent effect on the obesityphenotype. At the end of the study (29 weeks post-transplant and 36weeks post-diet) mice on the 45-60% fat diets (sham and treated) hadsignificantly higher body weight, adiposity (epididymal fat pad weightas a proportion of body weight) and circulating leptin levels than LFDshams (FIGS. 10D-10F). The obesity phenotype was more subtle in westerndiet mice during the first seven weeks (FIG. 1A) and by the end of thestudy there were no significant differences in body weight, adiposity orleptin levels between Western-fed mice (sham and tx) and LFD shamcontrols (FIGS. 10D-10F). All HFD groups had significantly higher liverweight (FIG. 10G) and evidence of cytoplasmic vacuolation, consistentwith dietary lipidosis in the liver (not shown) compared to LFDcontrols. Transplant recipients fed 45-60% fat diets had significantlyreduced liver weight relative to shams (not shown), although a pathologyassessment did not reveal differences in cytoplasmic vacuolation inH&E-stained liver sections (not shown). Similarly, vacuolation of renaltubular epithelium was observed in kidney sections from all HFD groups(consistent with dietary lipidosis) and there was no effect of celltransplantation on this phenotype. Other tissue pathologies (AdiposeTissue, Perirenal; Ileum; Skeletal Muscle; Cecum; Jejunum; Spleen;Colon; Kidney; Stomach, Glandular; Duodenum; Liver; Stomach,Nonglandular; Heart; Lung; and Testis) were consistent with spontaneousage- and sex-related events and considered to be unrelated to exposureto diets or cell transplants.

Example 6: Combined Treatment with Pancreatic Endocrine Precursor CellTransplants and an Antidiabetic Drug Improved Diet-Induced Obesity andGlucose Tolerance

Mice were placed on either the 10% fat control diet (D12450K; n=8) forthe duration of the study or 60% fat diet (D12492; n=64) for 6 weeks,followed by one of the following treatment regimens for the remainder ofthe study (n=16 per group): 1) 60% fat diet with no drug (D12492); 2)custom-made 60% fat diet containing rosiglitazone (18 mg/kg diet or ˜3mg/kg BW per day; Cayman Chemical™, Ann Arbor, Mich.; Research Diets™custom diet formulation D08121002); 3) custom-made 60% fat dietcontaining sitagliptin (4 g/kg diet or ˜750 mg/kg BW per day;sitagliptin phosphate monohydrate, BioVision Inc.™, Milpitas, Calif.;Research Diets™ custom diet formulation D08062502R); or 4) 60% fat diet(D12492) and metformin in drinking water (1.25 mg/mL or ˜250 mg/kg BWper day; 1,1-Dimethylbiguanide hydrochloride, Sigma-Aldrich™). Treatmentgroups are summarized in TABLE 2, above. Mice rapidly developedattributes of T2D following HFD administration (FIG. 11).

At the time of transplantation (one week after drug administration), allHFD-fed mice were significantly heavier than LFD controls (FIG. 5F).Weight loss was observed within the first two weeks followingtransplantation in HFD-fed mice on antidiabetic drugs (FIGS. 5C, 5D and5E). In contrast, no change in body weight was observed during this timein either HFD transplant recipients without drug treatment (FIG. 5B), orsham mice on any drug (FIGS. 5A-E). All transplant recipients receivingantidiabetic drugs had significantly lower body weight on day 75 (FIG.5F) and reduced epididymal fat pad weight (relative to body weight; FIG.5G) compared to sham mice, such that neither parameter was differentfrom LFD-fed sham controls. There was no effect of transplantation onbody weight (FIGS. 5B and F) or circulating leptin levels (FIG. 5H) inHFD-fed mice without drug treatment, although we did observe a reductionin relative epididymal fat pad weight in this cohort (FIG. 5G). Thecombination of a cell transplant with either metformin or sitagliptinresulted in significantly reduced circulating leptin levels compared totheir respective sham controls (FIG. 5G). There was no effect of thecell therapy on restoring leptin levels in the rosiglitazone group (FIG.5G).

It appears that there is some “synergy” with the combination therapy ofpancreatic progenitor cell transplant and T2D small molecule treatmentwith regards to body weight since neither treatment alone had any effecton body weight (see FIGS. 5A and 5B). Similarly, at 12 weekspost-transplant, neither treatment alone had any effect on glucosetolerance (see FIGS. 6A and 6B), whereas the combinations tested causedsignificant improvements in blood glucose and body weight. Therefore,the effect of combination is greater than the sum of the treatmentsalone. The only exception to this might be Sitagliptin, which on its ownshowed a mild reduction in glucose tolerance.

Fasting blood glucose levels were not affected by any of the combinationtherapies throughout the study duration (data not shown). At 12 weekspost-transplant, mice in all HFD sham groups were glucose intolerantcompared to LFD controls, regardless of drug treatment (FIG. 6A). Therewas no effect of the cell therapy on glucose tolerance at 12 weekspost-transplant in the HFD-fed mice without drug treatment (FIG. 6B) andlikewise, the combination with rosiglitazone was also ineffective atthis time (FIG. 6E). The cell therapy significantly improved glucosetolerance at 12 weeks post-transplant when combined with eithermetformin (FIG. 6C) or sitagliptin treatment (FIG. 6D). Glycemic controlduring an oral glucose challenge was indistinguishable between the LFDcontrols and HFD-fed mice receiving sitagliptin with the cell therapy,with the exception only of a marginally higher peak glucose level at 15minutes post-gavage (FIG. 6D). The improved glucose tolerance in celltransplant recipients from the metformin- and sitagliptin-treated micewas associated with significantly reduced fasting mouse C-peptide levelscompared to their respective sham controls at 16 weeks post-transplant(FIG. 6G), an effect that was not yet evident at 4 weeks (FIG. 6F). Theimprovements in glucose tolerance were not associated with differencesin the function of hESC-derived grafts. All transplant recipients showedrobust glucose-responsive human C-peptide secretion at 16 weeks andthere were no differences in human C-peptide levels between HFD-fed micetreated with different antidiabetic drugs (FIG. 6G).

Example 7: Comparison of Stage 4 (S4) and Stage 7 (S7) Cell Transplantsin Male and Female Mice

Stage 4 cells pancreatic progenitors (similar to those in the aboveexamples) were compared to Stage 7 cells, which are more differentiatedpancreatic endocrine cells, were both transplanted into normal, healthmale or female mice, on a normal diet. Following transplantation thebody weights of the mice were compared following a 4 hour fast (FIG. 12)and an overnight fast (FIG. 13). In these studies, mice receiving Stage7 cells tended to weigh less than mice receiving Stage 4 cells, whetherweighted after a 4 hr (FIG. 12) or overnight (FIG. 13) fast.Accordingly, some Stage 7 cells may be slightly preferable for reducingweight gain.

Example 8: Comparison of Stage 7 (S7) Cell Transplants with Human IsletCell Transplants in Mice

Stage 7 cells and human islet cells were compared in mice that wereeither diabetic (i.e. given STZ) or not (i.e. not given STZ) prior totransplantation, wherein body weight (FIG. 14A) and blood glucose (FIG.14B) were compared post transplant. However, there was not a controlthat was given STZ, but not transplanted. Animals that received the celltransplants tended to gain less weight than control animals (FIG. 14A).As shown in FIG. 14B, those animals that received transplants appearedmore able to regulate blood glucose post transplant similar to controlmice. However, the there was a greater time lag (i.e. ˜120 days post Tx)for S7 Tx mice as compared to Human Islet TX mice (i.e. ˜15 days postTx), as would be expected. It is important to note that S7 recipientshave normal glucose by ˜100 days, and soon there after are actuallylower than controls (hypoglycemic), similar to that of human isletrecipients. The blood glucose level achieved is hypoglycemic for themice, but since humans have naturally lower blood glucose levels andsince the cells being transplanted are human cells, the blood glucoselevels would not be considered hypoglycemic for humans. It is possiblethat the lower blood glucose reflects the fact that we are transplantinghuman cells, which have a lower set point for glucose levels than mice.Alternatively or in addition, it could also reflect the fact that anexcess numbers of cells were transplanted.

When the same animals studied in FIGS. 14A and 14B were compared formouse leptin levels (FIG. 15A) leptin was consistently lower in thetransplanted mice (either Stage 7 or human islet) than controls. Leptinis typically proportional to fat mass and when fat mass was measured byDEXA, both the human islet and Stage 7 cells had lower fat mass (FIG.15B). Similarly, when body composition was compared between controls,human islet Tx mice and Stage 7 cell Tx mice the TX mice consistentlyhad lower lean body mass (FIG. 16A), fat mass (FIG. 16B) and % body fat(FIG. 16C).

Similarly, FIGS. 17A-D show reduced fat weight when comparing Stage 7transplanted cells with either Human islet transplants or control cellsin perirenal tissues (FIG. 17C), epididymal tissues (FIG. 17D),mesenteric fat (FIG. 17E) and in all fat pads (FIG. 17F), which werecollected at sacrifice. (Note, for human islet n=5, which includes 4mice that got STZ and one that did not get STZ). Accordingly,transplantation of Stage 7 human pancreatic progenitor cells appear tobe more effective at reducing fat than human islet cells in diabeticmodel mice. In FIG. 17A, there is shown data for both left and rightkidneys and it is noted that the peri-renal fat around the left kidneyas compared to the right kidney (except for the control the “right”kidney is labeled either “Human Islets Tx” or “Stage 7 Tx”), and whenthe data is combined for the right and left kidney the bar on the graphis designated “both”. In mice that got either islets or stage 7 cells,cells were transplanted into the right kidney, which sees greater fataccumulation than the left kidney, except in the controls. It is likelythat we see more fat accumulation around the kidney into which the cellswere transplanted, because we speculate that there is local accumulationof insulin, and insulin is adipogenic.

Therefore, transplant of human islets and human Stage 7 cells mayproduce weight loss. There are some reports of patients with T1D whoreceive an islet transplant and subsequently lose weight. This has beenassumed to be due to the surgical procedure, immunosuppressive drugs,and/or life style changes. However, perhaps the weight loss is due tothe islet cells themselves. It has been found that both human islets anddifferentiated human stem cells produce the peptide Glucagon-likepeptide-1 (GLP-1). GLP-1 is an incretin (i.e. a metabolic hormone) thatis known to increase the amount of insulin released by pancreaticbeta-cells and may subsequently decrease blood glucose. Furthermore,when injected into animals or humans, GLP-1 can produce weight loss andis now used as a drug in patients with T2D. In these patients weightloss has been reported.

Also, when cells are transplanted in combination with the drugSitagliptin, the most weight loss was observed (see FIG. 5D).Sitagliptin is a known inhibitor of the enzyme dipeptidyl peptidase-4(DPP-4), which degrades and inactivates GLP-1. Accordingly, it ispossible that one mechanism by which the cells reduce weight gain is byproducing GLP-1, and this affect is enhanced by stabilizing the GLP-1with Sitagliptin.

Example 9: Comparison of Stage 7 (S7) Macro-Encapsulated CellTransplants in Mice

At the moment, encapsulation is proposed to be the best way to avoid animmune response in a clinically setting. However, this has yet to bedetermined as we are unable to experimentally confirm this in an animalmodel (the macro-encapsulation device we used, made by TheraCyte, doesnot prevent xenograft rejection). Early results released from ViaCytesuggest that their similar devices may be working in a patient. Of note,while the stage 4 progenitor cells have done well in these devices, itwas uncertain whether more mature differentiated cells (i.e. Stages >4)would survive. Indeed studies with fetal versus mature islets have shownfetal islets (akin to Stage 4 cells) do survive, mature and functionwithin TheraCyte devices while adult islets have poor survival (Lee, SH. et al. 2009). Therefore, one may have predicted that the moreislet-like cells from advanced differentiation protocols would also notwork well. However, recently we have obtained data (see FIGS. 19 and 20)that demonstrate that Stage 7 cells can indeed survive and functionwithin these macro-encapsulation devices.

While we have tested macro-encapsulation, others are investigatingmicro-encapsulation as an alternative strategy (see for example, Vegas,A J. et al. 2016). Aside from encapsulation, inducing immune tolerancehas also been explored as a strategy to avoid an immune response in vivo(Szot, G L. et al. 2015). It remains to be determined if this is aclinically relevant approach.

Weekly fasting blood glucose and body weight monitoring showed thatthere were no differences in between mean weight or glycemia of animalsin any groups (see FIGS. 18B and 18C). However, when comparing Stage 4and Stage 6 cell transplants in deviceless and kidney capsule (KC)implanted mice C-peptide levels were significantly higher in S6 KC cellswithin lo weeks of the transplant (see FIG. 18A).

The graft retrieval after transplant followed by immunohistochemicalanalysis showed the TheraCyte devices used in FIGS. 19 and 20 containedpredominantly what appeared to be endocrine cells (Synaptophysinpositive). The cells appeared to be functioning well with good glucoseresponsive C-peptide production.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. The word “comprising” isused herein as an open-ended term, substantially equivalent to thephrase “including, but not limited to”, and the word “comprises” has acorresponding meaning. As used herein, the singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a thing” includes more thanone such thing. Citation of references herein is not an admission thatsuch references are prior art to an embodiment of the present invention.The invention includes all embodiments and variations substantially ashereinbefore described and with reference to the examples and drawings.

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What is claimed is:
 1. A method for improving glycemic control in asubject, the method comprising: implanting a population of pancreaticendocrine progenitor cells into the subject, wherein the pancreaticendocrine progenitor cells mature in vivo to produce a populationcomprising pancreatic endocrine cells.
 2. The method of claim 1, whereinthe subject is a risk of developing type 2 diabetes (T2D).
 3. The methodof claim 1, wherein the subject has type 2 diabetes (T2D).
 4. The methodof claim 1, the method further comprises administering a therapeuticallyeffective amount of one or more small molecule anti-diabetic drugs tothe subject.
 5. The method of claim 3, the method further comprisesadministering a therapeutically effective amount of one or more smallmolecule anti-diabetic drugs to the subject.
 6. The method of claim 1,wherein the pancreatic endocrine progenitor cells mature in vivo toproduce a population comprising at least 2% pancreatic endocrine cells.7. The method of claim 4, wherein the one or more small moleculeanti-diabetic drugs are selected from the following: meglitinides;sulfonylureas; dipeptidyl-peptidase 4 (DPP-4) inhibitors; biguanides;thiazolidinediones; alpha-glucosidase inhibitors; sodium-glucosetransporter 2 (SGLT-2) inhibitors; and bile acid sequestrants.
 8. Themethod of claim 5, wherein the one or more small molecule anti-diabeticdrugs are selected from the following: meglitinides; sulfonylureas;dipeptidyl-peptidase 4 (DPP-4) inhibitors; biguanides;thiazolidinediones; alpha-glucosidase inhibitors; sodium-glucosetransporter 2 (SGLT-2) inhibitors; and bile acid sequestrants.
 9. Themethod of claim 4, wherein the anti-diabetic drug is selected from thegroup consisting of: sitagliptin; metformin; and rosiglitazone.
 10. Themethod of claim 5, wherein the anti-diabetic drug is selected from thegroup consisting of: sitagliptin; metformin; and rosiglitazone.
 11. Amethod for improving glycemic control in a subject, the methodcomprising: implanting a population of pancreatic endocrine progenitorcells into the subject, wherein the pancreatic endocrine progenitorcells mature in vivo to produce a population comprising pancreaticendocrine cells; and administering to the subject a therapeuticallyeffective amount of sitagliptin; metformin; or rosiglitazone.
 12. Themethod of claim 11, wherein the pancreatic endocrine progenitor cellsmature in vivo to produce a population comprising at least 2% pancreaticendocrine cells.
 14. The method of claim 11, wherein the subject is arisk of developing type 2 diabetes (T2D).
 15. The method of claim 11,wherein the subject has type 2 diabetes (T2D).